Tuberculosis compositions and methods of using the same

ABSTRACT

The present disclosure provides fusion proteins comprising  Mycobacterium tuberculosis  (Mtb) antigens, nucleic acid molecules encoding the same, vectors comprising nucleic acid molecules, compositions comprising the same, and methods of eliciting an immune response against tuberculosis.

FIELD

The present disclosure is directed, in part, to fusion proteinscomprising Mycobacterium tuberculosis (Mtb) antigens, nucleic acidmolecules encoding the same, vectors comprising nucleic acid molecules,compositions comprising the same, and methods of eliciting an immuneresponse against tuberculosis.

BACKGROUND

Tuberculosis (TB) is a global health problem resulting in 8 million newcases and 2 million deaths each year. The emergence of multi-drug andtotally-drug resistant strains of TB only makes this problem moresevere. The life cycle of Mtb has 3 stages. In the acute phase followinginitial infection the bacteria replicate in the host and virulencefactors are expressed, leading to the generation of an immune responseby the host. As the immune response begins to control the infection, theMtb enters a latent, asymptomatic state in which the bacteria becomenon-replicating and are encased in granulomas. The bacterium can persistin this latent state in infected individuals for many years, makingdiagnosis and treatment of disease difficult. In some cases, thebacteria are reactivated and begin replicating again, leading back tothe disease state. Reactivation can occur for numerous reasons,including immune suppression caused by diseases such as HIV, treatmentssuch as chemotherapy, or the weakening of the immune system due toaging. An estimated 2 billion people are latently infected with Mtbworldwide, and reactivation of latent Mtb accounts for most new cases ofactive TB disease. Reactivation is associated with inflammation,necrosis and cavitation of the lung, a process that results in drainingof the lesions into the bronchus. Aerosols generated when individualswith bronchial lesions cough causes dissemination of the Mtb organism touninfected, susceptible persons, and the transmission cycle is thusmaintained.

The only currently available vaccine against TB, Mycobacterium bovis(Bacille Calmette-Guérin) (BCG), was first introduced in 1921. BCG hasbeen widely utilized and while studies show that for some purposes BCGis effective (e.g. against disseminated TB), it is known to beineffective with respect to preventing the development, persistence andreactivation of latent TB. There is an ongoing need to develop improved,more effective vaccines against TB. In particular, there is a need todevelop vaccines that provide protection against the development,maintenance and/or reactivation of latent tuberculosis infection. Withthe availability of the entire genomic sequence of Mtb, and the toolsfor bioinformatic and experimental analysis of Mtb antigens, many newpotential Mtb vaccine candidates have been identified in recent years.These include antigens that are involved in acute infection, maintenanceof latency, or reactivation of Mtb. There are a range of deliverystrategies in clinical development that are comprised of combinations ofthese and other antigens that have been tested in animal models and arecurrently or will soon be in clinical trials.

While vaccines are often effective to immunize individualsprophylactically or therapeutically against pathogen infection or humandiseases, there is a need for improved vaccines. There is also a needfor compositions and methods that produce an enhanced immune response.Likewise, while some immunotherapeutics are useful to modulate immuneresponse in a patient, there remains a need for improvedimmunotherapeutic compositions and methods.

SUMMARY

This disclosure describes an antigen cassette (and specified variants)that can be used to create tuberculosis vaccines comprising specifiedMycobacterium tuberculosis (Mtb) antigens which are involved with 3identified stages of disease: 1) infection or acute infection, 2)latency or the latent state, and 3) resuscitation or reactivation ofactive disease. The disclosure also describes the strategic combinationof antigens which are incorporated into a variety of delivery platformsin such a way as to provide pathways to a matrix of matched combinationsof antigen delivery to obtain an optimized immune response. The subjectmatter described herein can be used as a prophylactic or therapeutic TBvaccine. The initial selection of antigens for inclusion into a usablecassette was based on a number of parameters including, for example, athorough review of the literature, expression data, responses by human Tcells, inclusion of human immunogenic regions, mouse protection studies,and conservation in sequence across most strains of TB with full genomesequences. Specific antigens were then probed to be sure they were ableto be expressed in a variety of systems (BCG, protein, viral vectors,nucleic acids), that they were immunogenic, and they could be made asfusions in proteins or other vectors to simplify downstreammanufacturing concerns. All of the selected antigens were then shown tobe immunogenic in mice, either when used alone, or in a variety ofcombinations, to arrive at the present application.

The constructs described herein have been integrated into a specifiedrange of delivery platforms that include the following classes (but notexhaustive) of representative delivery platforms: 1) viral vectordelivery systems, 2) recombinant BCG, 3) recombinant purified proteinfusions, 4) DNA plasmid vector systems, and 5) RNA vector systems. Thesedelivery platforms can be used either in a single platform alone or incombinations as matched antigen prime-boost approaches. In addition, theuse of these antigens in a single rBCG vector system, which isenvisioned to be used as an antigen matched prime for a boost with anyof the modalities above, including protein, viral vectors, nucleicacids, or others.

The present disclosure provides fusion proteins that comprise at leastthree Mycobacterium tuberculosis (Mtb) antigens, wherein the fusionprotein comprises: at least one acute Mtb antigen, at least one latentMtb antigen, and at least one resuscitation Mtb antigen; or at least twolatent Mtb antigens, and at least one resuscitation Mtb antigen.

The present disclosure also provides nucleic acid molecules encodingfusion proteins that comprise at least three Mycobacterium tuberculosis(Mtb) antigens, wherein the fusion protein comprises: at least one acuteMtb antigen, at least one latent Mtb antigen, and at least oneresuscitation Mtb antigen; or at least two latent Mtb antigens, and atleast one resuscitation Mtb antigen.

The present disclosure also provides: compositions comprising the fusionproteins and a pharmaceutically acceptable carrier; vectors encoding thefusion proteins; compositions comprising the vectors and apharmaceutically acceptable carrier; cells comprising the vectors;compositions comprising the cells and a pharmaceutically acceptablecarrier.

The present disclosure also provides compositions that comprise at leastthree Mycobacterium tuberculosis (Mtb) antigens, wherein the compositioncomprises: at least one acute Mtb antigen, at least one latent Mtbantigen, and at least one resuscitation Mtb antigen; or at least twolatent Mtb antigens, and at least one resuscitation Mtb antigen.

The present disclosure also provides compositions that comprise at leastthree Mycobacterium tuberculosis (Mtb) antigens, wherein the compositioncomprises: at least one acute Mtb antigen, at least one latent Mtbantigen, and at least one resuscitation Mtb antigen; or at least twolatent Mtb antigens, and at least one resuscitation Mtb antigen; whereinthe composition comprises at least one nucleic acid molecule encoding atleast one of the Mtb antigens.

The present disclosure also provides methods of eliciting an immuneresponse against Mycobacterium tuberculosis in a mammal comprisingadministering to the mammal an immunologically sufficient amount of oneor more fusion proteins comprising at least three Mycobacteriumtuberculosis (Mtb) antigens, wherein at least one fusion proteincomprises: at least one acute Mtb antigen, at least one latent Mtbantigen, and at least one resuscitation Mtb antigen; or at least twolatent Mtb antigens, and at least one resuscitation Mtb antigen.

The present disclosure also provides methods of eliciting an immuneresponse against Mycobacterium tuberculosis in a mammal comprisingadministering to the mammal an immunologically sufficient amount of acomposition comprising at least three Mycobacterium tuberculosis (Mtb)antigens, wherein the composition comprises: at least one acute Mtbantigen, at least one latent Mtb antigen, and at least one resuscitationMtb antigen; or at least two latent Mtb antigens, and at least oneresuscitation Mtb antigen; and a pharmaceutically acceptable carrier.

The present disclosure also provides methods of eliciting an immuneresponse against Mycobacterium tuberculosis in a mammal comprisingadministering to the mammal an immunologically sufficient amount of acomposition comprising at least three Mycobacterium tuberculosis (Mtb)antigens, wherein the composition comprises: at least one acute Mtbantigen, at least one latent Mtb antigen, and at least one resuscitationMtb antigen; or at least two latent Mtb antigens, and at least oneresuscitation Mtb antigen; and a pharmaceutically acceptable carrier;wherein the composition comprises at least one nucleic acid moleculeencoding at least one of the Mtb antigens.

The present disclosure also provides fusion proteins for use in thepreparation of a medicament for treating or preventing a Mycobacteriumtuberculosis infection, wherein the fusion protein comprises at leastthree Mycobacterium tuberculosis (Mtb) antigens, and wherein the fusionprotein comprises: at least one acute Mtb antigen, at least one latentMtb antigen, and at least one resuscitation Mtb antigen; or at least twolatent Mtb antigens, and at least one resuscitation Mtb antigen.

The present disclosure also provides fusion proteins for use in treatingor preventing a Mycobacterium tuberculosis infection, wherein the fusionprotein comprises at least three Mycobacterium tuberculosis (Mtb)antigens, and wherein the fusion protein comprises: at least one acuteMtb antigen, at least one latent Mtb antigen, and at least oneresuscitation Mtb antigen; or at least two latent Mtb antigens, and atleast one resuscitation Mtb antigen.

The present disclosure also provides uses of a fusion protein in thepreparation of a medicament for treating or preventing a Mycobacteriumtuberculosis infection, wherein the fusion protein comprises at leastthree Mycobacterium tuberculosis (Mtb) antigens, and wherein the fusionprotein comprises: at least one acute Mtb antigen, at least one latentMtb antigen, and at least one resuscitation Mtb antigen; or at least twolatent Mtb antigens, and at least one resuscitation Mtb antigen.

The present disclosure also provides uses of a fusion protein intreating or preventing a Mycobacterium tuberculosis infection, whereinthe fusion protein comprises at least three Mycobacterium tuberculosis(Mtb) antigens, and wherein the fusion protein comprises: at least oneacute Mtb antigen, at least one latent Mtb antigen, and at least oneresuscitation Mtb antigen; or at least two latent Mtb antigens, and atleast one resuscitation Mtb antigen.

The present disclosure also provides compositions for use in thepreparation of a medicament for treating or preventing a Mycobacteriumtuberculosis infection, wherein the composition comprises at least threeMycobacterium tuberculosis (Mtb) antigens, and wherein the compositioncomprises: at least one acute Mtb antigen, at least one latent Mtbantigen, and at least one resuscitation Mtb antigen; or at least twolatent Mtb antigens, and at least one resuscitation Mtb antigen; and apharmaceutically acceptable carrier.

The present disclosure also provides composition for use in treating orpreventing a Mycobacterium tuberculosis infection, wherein thecomposition comprises at least three Mycobacterium tuberculosis (Mtb)antigens, and wherein the composition comprises: at least one acute Mtbantigen, at least one latent Mtb antigen, and at least one resuscitationMtb antigen; or at least two latent Mtb antigens, and at least oneresuscitation Mtb antigen; and a pharmaceutically acceptable carrier.

The present disclosure also provides uses of a composition in thepreparation of a medicament for treating or preventing a Mycobacteriumtuberculosis infection, wherein the composition comprises at least threeMycobacterium tuberculosis (Mtb) antigens, and wherein the compositioncomprises: at least one acute Mtb antigen, at least one latent Mtbantigen, and at least one resuscitation Mtb antigen; or at least twolatent Mtb antigens, and at least one resuscitation Mtb antigen; and apharmaceutically acceptable carrier.

The present disclosure also provides uses of a composition in treatingor preventing a Mycobacterium tuberculosis infection, wherein thecomposition comprises at least three Mycobacterium tuberculosis (Mtb)antigens, and wherein the composition comprises: at least one acute Mtbantigen, at least one latent Mtb antigen, and at least one resuscitationMtb antigen; or at least two latent Mtb antigens, and at least oneresuscitation Mtb antigen; and a pharmaceutically acceptable carrier.

The present disclosure also provides compositions for use in thepreparation of a medicament for treating or preventing a Mycobacteriumtuberculosis infection, wherein the composition comprises at least threeMycobacterium tuberculosis (Mtb) antigens, and wherein the compositioncomprises: at least one acute Mtb antigen, at least one latent Mtbantigen, and at least one resuscitation Mtb antigen; or at least twolatent Mtb antigens, and at least one resuscitation Mtb antigen; and apharmaceutically acceptable carrier; wherein the composition comprisesat least one nucleic acid molecule encoding at least one of the Mtbantigens.

The present disclosure also provides compositions for use in treating orpreventing a Mycobacterium tuberculosis infection, wherein thecomposition comprises at least three Mycobacterium tuberculosis (Mtb)antigens, and wherein the composition comprises: at least one acute Mtbantigen, at least one latent Mtb antigen, and at least one resuscitationMtb antigen; or at least two latent Mtb antigens, and at least oneresuscitation Mtb antigen; and a pharmaceutically acceptable carrier;wherein the composition comprises at least one nucleic acid moleculeencoding at least one of the Mtb antigens.

The present disclosure also provides uses of a composition in thepreparation of a medicament for treating or preventing a Mycobacteriumtuberculosis infection, wherein the composition comprises at least threeMycobacterium tuberculosis (Mtb) antigens, and wherein the compositioncomprises: at least one acute Mtb antigen, at least one latent Mtbantigen, and at least one resuscitation Mtb antigen; or at least twolatent Mtb antigens, and at least one resuscitation Mtb antigen; and apharmaceutically acceptable carrier; wherein the composition comprisesat least one nucleic acid molecule encoding at least one of the Mtbantigens.

The present disclosure also provides uses of a composition in treatingor preventing a Mycobacterium tuberculosis infection, wherein thecomposition comprises at least three Mycobacterium tuberculosis (Mtb)antigens, and wherein the composition comprises: at least one acute Mtbantigen, at least one latent Mtb antigen, and at least one resuscitationMtb antigen; or at least two latent Mtb antigens, and at least oneresuscitation Mtb antigen; and a pharmaceutically acceptable carrier;wherein the composition comprises at least one nucleic acid moleculeencoding at least one of the Mtb antigens.

The present disclosure also provides fusion proteins, compositions,cells, vectors, methods, and uses, as described herein, substantially asdescribed with reference to the accompanying examples and/or figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show maps of the plasmids used to insert the genes ofinterest into the chromosome of BCG SSI or other strains of BCG: (A)constructs with the Ag85B signal sequence for secretion of the fusions;and (B) constructs with the 19 kDa signal sequence to anchor the fusionsinto the membrane.

FIG. 2 shows in vitro antigen responsiveness after vaccination with BCGstrains carrying antigen cassette.

FIGS. 3A and 3B show in vitro stimulation: (A) showing INF-γ inductionin splenocytes following protein stimulation and ELISpot; and (B)analysis showing number of splenocytes expressing INF-γ from CB6F1 miceimmunized twice with 10 μg of the 5 Ag fusion protein (Construct D) anda synthetic poly I:C adjuvant.

FIGS. 4A and 4B show in vitro stimulation: (A) ELISpot; and (B) analysisof splenocytes from CB6F1 mice immunized twice with 3 μg of the 5 Agfusion protein (Construct D) or 4 Ag fusion protein (Construct A) and asynthetic MPL TLR4 adjuvant; when Ag85B is removed from the 5 Ag fusionprotein, the responses to the other 4 antigens in the 4 Ag proteinincrease.

FIGS. 5A and 5B show in vitro stimulation: (A) ELISpot; and (B) analysisof splenocytes from CB6F1 mice immunized twice with 3 μg of the 5 Agfusion protein (Construct D) and 4 Ag fusion proteins with eitherwild-type or modified Rv1733 or the 4 Ag protein with RpfD replaced byRpfB, with RpfB either at the 3′ or 5′ end of the fusion protein; theproteins were adjuvanted with a synthetic poly I:C adjuvant; RpfBinduces a much stronger immune response than RpfD, particularly whenRpfB is at the 5′ end of the fusion protein; neither modified norwild-type Rv1733 induced a strong immune response.

FIGS. 6A and 6B show bacterial numbers in: (A) the lungs and (B) spleenof CB6F1 mice primed with BCG and boosted with either the 4 Ag or 5 Agfusion protein 4 weeks after challenge with Mycobacterium tuberculosisH37Rv.

DESCRIPTION OF EMBODIMENTS

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting.

As used herein, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise.

For recitation of numeric ranges herein, each intervening number therebetween with the same degree of precision is explicitly contemplated.For example, for the range of 6-9, the numbers 7 and 8 are contemplatedin addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitlycontemplated.

As used herein, “acute Mtb antigen” means any Mtb antigen involved inthe acute phase tuberculosis infection.

As used herein, “adjuvant” means any molecule added to any compositiondescribed herein to enhance the immunogenicity of the Mtb antigens.

As used herein, “coding sequence” or “encoding nucleic acid” means thenucleic acids (RNA or DNA molecule) that comprise a nucleotide sequencewhich encodes an Mtb antigen. The coding sequence can further includeinitiation and termination signals operably linked to regulatoryelements including a promoter and polyadenylation signal capable ofdirecting expression in the cells of an individual or mammal to whichthe nucleic acid is administered.

As used herein, “consensus” or “consensus sequence” means a polypeptidesequence based on analysis of an alignment of multiple subtypes of aparticular Mtb antigen. Nucleic acid sequences that encode a consensuspolypeptide sequence can be prepared. Vaccines comprising Mtb antigensthat comprise consensus sequences and/or nucleic acid molecules thatencode such antigens can be used to induce broad immunity againstmultiple subtypes or serotypes of a particular antigen.

As used herein, “electroporation” means the use of a transmembraneelectric field pulse to induce microscopic pathways (pores) in abio-membrane; their presence allows biomolecules such as plasmids,oligonucleotides, siRNA, drugs, ions, and water to pass from one side ofthe cellular membrane to the other.

As used herein, “fragment” with respect to nucleic acid sequences, meansa nucleic acid sequence or a portion thereof, that encodes a portion ofan Mtb antigen capable of eliciting an immune response in a mammal thatcross reacts with a full length wild type Mtb antigen. The fragments canbe DNA fragments selected from at least one of the various nucleotidesequences that encode protein fragments set forth below.

As used herein, “fragment” or “immunogenic fragment” with respect topolypeptide sequences, means a portion of an MTB antigen capable ofeliciting an immune response in a mammal that cross reacts with a fulllength wild type strain Mtb antigen. Fragments of consensus or wild typeMtb antigens can comprise at least 10%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90% or at least 95% of a consensus or wild type Mtb antigen. Insome embodiments, fragments of consensus proteins can comprise at least20 amino acids or more, at least 30 amino acids or more, at least 40amino acids or more, at least 50 amino acids or more, at least 60 aminoacids or more, at least 70 amino acids or more, at least 80 amino acidsor more, at least 90 amino acids or more, at least 100 amino acids ormore, at least 110 amino acids or more, at least 120 amino acids ormore, at least 130 amino acids or more, at least 140 amino acids ormore, at least 150 amino acids or more, at least 160 amino acids ormore, at least 170 amino acids or more, at least 180 amino acids or moreof a consensus or wild type protein.

As used herein, “genetic construct” refers to the DNA or RNA moleculesthat comprise a nucleotide sequence which encodes an Mtb antigen. Thecoding sequence includes initiation and termination signals operablylinked to regulatory elements including a promoter and polyadenylationsignal capable of directing expression in the cells of the individual towhom the nucleic acid molecule is administered.

As used herein, “expressible form” refers to gene constructs thatcontain the necessary regulatory elements operable linked to a codingsequence that encodes an Mtb antigen such that when present in the cellof the individual, the coding sequence will be expressed.

As used herein, “homology” refers to a degree of complementarity. Therecan be partial homology or complete homology (i.e., identity). Apartially complementary sequence that at least partially inhibits acompletely complementary sequence from hybridizing to a target nucleicacid is referred to using the functional term “substantiallyhomologous.” When used in reference to a double-stranded nucleic acidsequence such as a cDNA or genomic clone, the term “substantiallyhomologous” refers to a probe that can hybridize to a strand of thedouble-stranded nucleic acid sequence under conditions of lowstringency. When used in reference to a single-stranded nucleic acidsequence, the term “substantially homologous” refers to a probe that canhybridize to (i.e., is the complement of) the single-stranded nucleicacid template sequence under conditions of low stringency.

As used herein, “identical” or “identity” in the context of two or morenucleic acids or polypeptide sequences, means that the sequences have aspecified percentage of residues that are the same over a specifiedregion. The percentage can be calculated by optimally aligning the twosequences, comparing the two sequences over the specified region,determining the number of positions at which the identical residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the specified region, and multiplying the result by 100 toyield the percentage of sequence identity. In cases where the twosequences are of different lengths or the alignment produces one or morestaggered ends and the specified region of comparison includes only asingle sequence, the residues of single sequence are included in thedenominator but not the numerator of the calculation. When comparing DNAand RNA, thymine (T) and uracil (U) residues can be consideredequivalent. Identity can be performed manually or by using a computersequence algorithm such as BLAST or BLAST 2.0.

As used herein, “immune response” means the activation of a host'simmune system, e.g., that of a mammal, in response to the introductionof an Mtb antigen. The immune response can be in the form of a cellularor humoral response, or both.

As used herein, “latent Mtb antigen” means any Mtb antigen involved inthe latent phase tuberculosis infection.

As used herein, “Mtb antigen” means an antigen from Mycobacteriumtuberculosis, which may be an isolated antigen, or an antigen that formspart of a fusion protein with other antigen(s).

As used herein, “nucleic acid” or “oligonucleotide” or “polynucleotide”means at least two nucleotides covalently linked together. The depictionof a single strand also defines the sequence of the complementarystrand. Thus, a nucleic acid also encompasses the complementary strandof a depicted single strand. Many variants of a nucleic acid can be usedfor the same purpose as a given nucleic acid. Thus, a nucleic acid alsoencompasses substantially identical nucleic acids and complementsthereof. A single strand provides a probe that can hybridize to a targetsequence under stringent hybridization conditions. Thus, a nucleic acidalso encompasses a probe that hybridizes under stringent hybridizationconditions. Nucleic acids can be single stranded or double stranded, orcan contain portions of both double stranded and single strandedsequence. The nucleic acid can be DNA, both genomic and cDNA, RNA, or ahybrid, where the nucleic acid can contain combinations of deoxyribo-and ribo-nucleotides, and combinations of bases including uracil,adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine,isocytosine and isoguanine. Nucleic acids can be obtained by chemicalsynthesis methods or by recombinant methods.

As used herein, “operably linked” means that expression of a gene isunder the control of a promoter with which it is spatially connected. Apromoter can be positioned 5′ (upstream) or 3′ (downstream) of a geneunder its control. The distance between the promoter and a gene can beapproximately the same as the distance between that promoter and thegene it controls in the gene from which the promoter is derived. As isknown in the art, variation in this distance can be accommodated withoutloss of promoter function.

As used herein, “promoter” means a synthetic or naturally-derivedmolecule which is capable of conferring, activating or enhancingexpression of a nucleic acid in a cell. A promoter can comprise one ormore specific transcriptional regulatory sequences to further enhanceexpression and/or to alter the spatial expression and/or temporalexpression of same. A promoter can also comprise distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription. A promoter can bederived from sources including viral, bacterial, fungal, plants,insects, and animals. A promoter can regulate the expression of a genecomponent constitutively, or differentially with respect to cell, thetissue or organ in which expression occurs or, with respect to thedevelopmental stage at which expression occurs, or in response toexternal stimuli such as physiological stresses, pathogens, metal ions,or inducing agents.

As used herein, “resuscitation Mtb antigen” means any Mtb antigeninvolved in the resuscitation or reactivation of a tuberculosisinfection.

As used herein, “signal peptide” and “leader sequence”, usedinterchangeably, refer to an amino acid sequence that can be linked atthe amino terminus of an Mtb antigenic protein set forth herein. Signalpeptides/leader sequences typically direct localization of a protein.Signal peptides/leader sequences used herein can facilitate secretion ofthe protein from the cell in which it is produced or anchor it in themembrane. Signal peptides/leader sequences are often cleaved from theremainder of the protein, often referred to as the mature protein, uponsecretion from the cell. Signal peptides/leader sequences are linked atthe N terminus of the protein.

As used herein, “stringent hybridization conditions” means conditionsunder which a first nucleic acid sequence (e.g., probe) will hybridizeto a second nucleic acid sequence (e.g., target), such as in a complexmixture of nucleic acids. Stringent conditions are sequence-dependentand will be different in different circumstances. Stringent conditionscan be selected to be about 5 to 10° C. lower than the thermal meltingpoint (T_(m)) for the specific sequence at a defined ionic strength pH.The T_(m) can be the temperature (under defined ionic strength, pH, andnucleic concentration) at which 50% of the probes complementary to thetarget hybridize to the target sequence at equilibrium (as the targetsequences are present in excess, at T_(m), 50% of the probes areoccupied at equilibrium). Stringent conditions can be those in which thesalt concentration is less than about 1.0 M sodium ion, such as about0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3and the temperature is at least about 30° C. for short probes (e.g.,about 10 to 50 nucleotides) and at least about 60° C. for long probes(e.g., greater than about 50 nucleotides). Stringent conditions can alsobe achieved with the addition of destabilizing agents such as formamide.For selective or specific hybridization, a positive signal can be atleast 2 to 10 times background hybridization. Exemplary stringenthybridization conditions include the following: 50% formamide, 5×SSC,and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS, incubating at 65°C., with wash in 0.2×SSC, and 0.1% SDS at 65° C.

As used herein, “substantially complementary” means that a firstsequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or99% identical to the complement of a second sequence over a region of 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360,450, 540, or more nucleotides or amino acids, or that the two sequenceshybridize under stringent hybridization conditions.

As used herein, “substantially identical” means that a first and secondsequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%or 99% identical over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100, 180, 270, 360, 450, 540 or more nucleotides oramino acids, or with respect to nucleic acids, if the first sequence issubstantially complementary to the complement of the second sequence.

As used herein, “variant” with respect to a nucleic acid means: i) aportion or fragment of a referenced nucleotide sequence; ii) thecomplement of a referenced nucleotide sequence or portion thereof; iii)a nucleic acid that is substantially identical to a referenced nucleicacid or the complement thereof; or iv) a nucleic acid that hybridizesunder stringent conditions to the referenced nucleic acid, complementthereof, or a sequences substantially identical thereto.

As used herein, “variant” with respect to a peptide or polypeptide meansthat it differs in amino acid sequence by the insertion, deletion, orconservative substitution of amino acids, but retains at least onebiological activity. Variant can also mean a protein with an amino acidsequence that is substantially identical to a referenced protein with anamino acid sequence that retains at least one biological activity. Aconservative substitution of an amino acid, i.e., replacing an aminoacid with a different amino acid of similar properties (e.g.,hydrophilicity, degree and distribution of charged regions) isrecognized in the art as typically involving a minor change Amino acidsubstitutions that are compatible with biological function areunderstood to depend on the relative similarity of the amino acids, andparticularly the side chains of those amino acids, as revealed by thehydrophobicity, hydrophilicity, charge, size, and other properties.

As used herein, “vector” means a nucleic acid sequence containing anorigin of replication. A vector can be a viral vector, bacteriophage,bacterial artificial chromosome or yeast artificial chromosome. A vectorcan be a DNA or RNA vector. A vector can be a self-replicatingextrachromosomal vector.

The present disclosure provides fusion proteins comprising at leastthree Mycobacterium tuberculosis (Mtb) antigens. In some embodiments,the fusion protein comprises at least one acute Mtb antigen, at leastone latent Mtb antigen, and at least one resuscitation Mtb antigen. Insome embodiments, the fusion protein comprises at least two latent Mtbantigens and at least one resuscitation Mtb antigen.

In some embodiments, the nucleic acid molecule encoding any particularMtb antigen can be a mycobacterial sequence, a bacterial codon optimizedsequence (such as an E. coli optimized sequence), or a mammalianoptimized sequence (such as a human optimized sequence). The E. colioptimized sequences can be used, for example, to produce fusionproteins. The human optimized sequences can be used in, for example,viral vectors. Methods of codon optimization (whether for bacterial ormammalian) are well known to the skilled artisan.

In some embodiments, the acute Mtb antigen is Ag85B, ESAT6, MPT64,PPE15, PPE51, or Rv3615c. In some embodiments, the acute Mtb antigen isAg85B, ESAT6, or Rv3615c. In some embodiments, the acute Mtb antigen isAg85B or ESAT6. Additional acute Mtb antigens are well known to theskilled artisan.

The acute Mtb antigen Ag85B is also known as Rv1886c. A nucleotidesequence encoding Ag85B is shown in Table 1 as SEQ ID NO:1(mycobacterial sequence; not codon optimized), SEQ ID NO:2 (E. colioptimized), and SEQ ID NO:3 (human optimized), and an amino acidsequence of Ag85B is shown in Table 1 as SEQ ID NO:4 (mycobacterialsequence) and SEQ ID NO:5 (E. coli and human optimized).

The acute Mtb antigen ESAT-6 is also known as Rv3875. A nucleotidesequence encoding ESAT-6 is shown in Table 1 as SEQ ID NO:6(mycobacterial sequence; not codon optimized) and SEQ ID NO:7 (humanoptimized), and an amino acid sequence of ESAT-6 is shown in Table 1 asSEQ ID NO:8.

The acute Mtb antigen MPT64 is also known as Rv1980c. A nucleotidesequence encoding the acute Mtb antigen MPT64 is shown in Table 1 as SEQID NO:9 (mycobacterial sequence; not codon optimized) and as SEQ IDNO:10 (human optimized), and an amino acid sequence of MPT64 is shown inTable 1 as SEQ ID NO:11.

The acute Mtb antigen PPE15 is also known as Rv1039c. A nucleotidesequence encoding the acute Mtb antigen PPE15 is shown in Table 1 as SEQID NO:12 (mycobacterial sequence; not codon optimized) and as SEQ IDNO:13 (human optimized), and an amino acid sequence of PPE15 is shown inTable 1 as SEQ ID NO:14.

The acute Mtb antigen PPE51 is also known as Rv3136. A nucleotidesequence encoding the acute Mtb antigen PPE51 is shown in Table 1 as SEQID NO:15 (mycobacterial sequence; not codon optimized), SEQ ID NO:16 (E.coli optimized) and as SEQ ID NO:17 (human optimized), and an amino acidsequence of PPE51 is shown in Table 1 as SEQ ID NO:18.

A nucleotide sequence encoding the acute Mtb antigen Rv3615c is shown inTable 1 as SEQ ID NO:19 (mycobacterial sequence; not codon optimized)and as SEQ ID NO:20 (human optimized), and an amino acid sequence ofRv3615c is shown in Table 1 as SEQ ID NO:21.

TABLE 1 nucleotide sequence Construct amino acid sequence Ag85Batgacagacgtgagccgaaagattcgagcttggggacgccgattgatgatcggcacggcagcggctgtagtccttccgggcctggtggggcttgccgcggagcggcaaccgcgggcgcgttctcccggccggggctgccggtcgagtacctgcaggtgccgtcgccgtcgatgggccgcgacatcaaggttcagttccagagcggtgggaacaactcacctgeggtttatctgetcgacggcctgcgcgcccaagacgactacaacggctgggatatcaacaccceggcgttcgagtggtactaccagtegggactgtegatagtcatgccggteggegggcagtccagettctacagcgactggtacagcccggcctgcggtaaggctggctgccagacttacaagtgggaaaccttcctgaccagcgagctgccgcaatggttgtccgccaacagggccgtgaagcccaccggcagcgctgcaatcggcttgtcgatggccggctcgtcggcaatgatcttggccgcctaccacccccagcagttcatctacgccggctcgctgtcggccctgctggacccctctcaggggatggggcctagcctgatcggcctcgcgatgggtgacgccggcggttacaaggccgcagacatgtggggtccctcgagtgacccggcatgggagcgcaacgaccctacgcagcagatccccaagctggtcgcaaacaacacccggctatgggtttattgegggaacggcaccccgaacgagttgggcggtgccaacatacccgccgagttcttggagaacttcgttcgtagcagcaacctgaagttccaggatgcgtacaacgccgcgggegggcacaacgccgtgttcaacttcccgcccaacggcacgcacagctgggagtactggggcgctcagctcaacgccatgaagggtgacctgcagagttcgttaggcgccggctga (SEQ ID NO: 1)atgtttagccgtcctggcctgccagttgaatacctgcaagttccgagcccgtccatgggtcgtgacattaaggtgcagttccagagcggcggtaacaatagcccggctgtgtacctgctggacggtctgcgtgcgcaggatgattacaacggctgggacatcaataccccggcatttgagtggtattaccagtcgggtctgagcattgtgatgccggttggcggtcaaagcagcttctatagcgattggtacagcccggcatgcggcaaggctggttgccaaacctacaagtgggaaactttatgaccagcgagctgccgcaatggttgagcgccaaccgtgcggtcaaaccgaccggtagcgctgctattggcctgtccatggccggcagcagcgcgatgatcttggcggcataccatccgcagcagtttatctacgccggtagcctgagcgcattgctggacccgagccaaggcatgggtccgagcctgattggtctggcaatgggtgacgcaggtggttacaaagcggccgatatgtggggcccatctagcgacccggcatgggagcgtaatgacccgacccagcaaattccgaaactggtggcgaataacacgcgcctgtgggtctactgtggcaatggtacgccgaacgagctgggtggcgcgaatatccctgcggagtttctggaaaactttgttcgcagcagcaacctgaaattccaggacgcgtataacgcagccggtggtcacaatgeggttttcaatttcccgccaaatggcactcatagctgggagtactggggtgcgcagttgaacgcaatgaaaggcgatctgcaatcctctctgggtgegggc (SEQ ID NO: 2)atgttctccaggcccggcctgcctgtcgagtatctgcaggtcccctccccctccatgggcagagacatcaaggtgcagttccaatccggaggcaacaacagccccgccgtgtatctectcgacggcctgagggctcaggacgactacaacggctgggacatcaacacccccgccttcgagtggtactaccagtccggactgagcatcgtcatgcccgtgggcggccagagctccttctacagcgactggtatagccctgcctgcggcaaagccggatgccagacctacaagtgggagacctttctgaccagcgaactgccccagtggctgtccgccaatagggccgtcaaacctaccggctccgctgccatcggactcagcatggccggaagctccgctatgatcctggccgcctaccacccccagcaatttatctacgctggcagcctgtccgctctgctggatcctagccaaggcatgggccctagcctcattggcctggccatgggcgatgctggcggctataaggccgccgatatgtggggccctagctccgatcctgcctgggagaggaatgaccccacccagcagatccccaagctggtggccaacaacacaaggctctgggtgtactgeggcaatggcacccccaacgaactgggeggagccaacattcccgccgagttcctggagaacttcgtcaggagcagcaacctgaagttccaggacgcctacaatgccgccggaggccacaacgctgtgttcaacttccctcccaacggcacccacagctgggagtattggggcgctcagctgaacgccatgaaaggcgacctccagagctccctgggagctgga (SEQ ID NO: 3)MTDVSRKIRAWGRRLMIGTAAAVVLPGLVGLAGGAATAGAFSRPGLPVEYLQVPSPSMGRDIKVQFQSGGNNSPAVYLLDGLRAQDDYNGWDINTPAFEWYYQSGLSIVMPVGGQSSFYSDWYSPACGKAGCQTYKWETFLTSELPQWLSANRAVKPTGSAAIGLSMAGSSAMILAAYHPQQFIYAGSLSALLDPSQGMGPSLIGLAMGDAGGYKAADMWGPSSDPAWERNDPTQQIPKLVANNTRLWVYCGNGTPNELGGANIPAEFLENFVRSSNLKFQDAYNAAGGHNAVFNFPPNGTHSWEYWGAQLNAMKGDLQSSLGAG (SEQ ID NO: 4)MFSRPGLPVEYLQVPSPSMGRDIKVQFQSGGNNSPAVYLLDGLRAQDDYNGWDINTPAFEWYYQSGLSIVMPVGGQSSFYSDWYSPACGKAGCQTYKWETFLTSELPQWLSANRAVKPTGSAAIGLSMAGSSAMILAAYHPQQFIYAGSLSALLDPSQGMGPSLIGLAMGDAGGYKAADMWGPSSDPAWERNDPTQQIPKLVANNTRLWVYCGNGTPNELGGANIPAEFLENFVRSSNLKFQDAYNAAGGHNAVFNFPPNGTHSWEYWGAQLNAMKGDLQSSLGAG (SEQ ID NO: 5) ESAT6atgacagagcagcagtggaatttcgcgggtatcgaggccgcggcaagcgcaatccagggaaatgtcacgtccattcattccctecttgacgaggggaagcagtccctgaccaagctcgcagcggcctggggcggtagcggttcggaggcgtaccagggtgtccagcaaaaatgggacgccacggctaccgagctgaacaacgcgctgcagaacctggcgcggacgateagcgaagccggtcaggcaatggatcgaccgaaggcaacgtcactgggatgttcgcatag (SEQ ID NO: 6)accgagcagcagtggaacttcgccggcatcgaagctgccgctagcgccatccaaggcaacgtgaccagcatccacagcctgctggacgagggcaagcagagcctgaccaagctggctgctgcttggggcggatccggaagcgaagcctaccagggcgtgcagcagaagtgggacgccacagccaccgagctgaacaacgccctgcagaacctcgccagaaccatcagcgaggccggacaggctatggccagcacagagggcaatgtgaccggcatgttcgcc (SEQ ID NO: 7)TEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWDATATELNNALQNLARTISEAGQAMASTEGNVTGMFA (SEQ ID NO: 8) MPT64gtgcgcatcaagatcttcatgctggtcacggctgtcgttttgctctgttgttcgggtgtggccacggccgcgcccaagacctactgcgaggagttgaaaggcaccgataccggccaggcgtgccagattcaaatgtccgacccggcctacaacatcaacatcagcctgcccagttactaccccgaccagaagtcgctggaaaattacatcgcccagacgcgcgacaagttectcagcgcggccacatcgtccactccacgcgaagccccctacgaattgaatatcacctcggccacataccagtccgcgataccgccgcgtggtacgcaggccgtggtgctcaaggtctaccagaacgccggcggcacgcacccaacgaccacgtacaaggccttcgattgggaccaggcctatcgcaagccaatcacctatgacacgctgtggcaggctgacaccgatccgctgccagtcgtcttccccattgtgcaaggtgaactgagcaagcagaccggacaacaggtatcgatagcgccgaatgccggcttggacccggtgaattatcagaacttcgcagtcacgaacgacggggtgattttatcttcaacccgggggagttgctgcccgaagcagccggcccaacccaggtattggtcccacgttccgcgatcgactcgatgctggcctag (SEQ ID NO: 9)atggtcaggatcaagatcttcatgctcgtgaccgccgtggtgctcctgtgttgttccggcgtggctaccgctgctcccaagacctactgcgaggagctgaagggaaccgacaccggccaggcctgccagatccaaatgagcgaccccgcctacaacatcaacatctccctcccctcctactaccccgateagaagtccctcgagaactacatcgctcagaccagggacaagttectgagcgccgccacaagcagcacacccagagaggccccctacgagctgaacatcacctccgccacctaccagtccgctattectcccagaggcacccaggctgtggtgctcaaggtctaccaaaacgctggeggaacacaccccaccaccacctacaaggccttcgactgggaccaggcctacaggaagcccatcacatacgacaccctgtggcaggctgataccgatccectgcccgtggtgttccccatcgtgcagggcgagctctccaagcagaccggccagcaagtgagcatcgcccccaatgctggactggaccccgtgaactaccagaacttcgccgtcaccaacgacggcgtgatcttcttcttcaatcccggcgaactgctgcctgaagctgctggccccacccaagtgctggtgcctagaagcgccatcgactccatgctggcctga(SEQ ID NO: 10)VRIKIFMLVTAVVLLCCSGVATAAPKTYCEELKGTDTGQACQIQMSDPAYNINISLPSYYPDQKSLENYIAQTRDKFLSAATSSTPREAPYELNITSATYQSAIPPRGTQAVVLKVYQNAGGTHPTTTYKAFDWDQAYRKPITYDTLWQADTDPLPVVFPIVQGELSKQTGQQVSIAPNAGLDPVNYQNFAVTNDGVIFFFNPGELLPEAAGPTQVLVPRSAIDSMLA (SEQ ID NO: 11) PPE15atggattteggagattaccccctgagatcaactccgcacgcatgtacgccggcgcgggtgcaggaccgatgatggccgccggggccgcatggaacggcctggccgccgagttgggtacgacggccgcgtcgtatgagtcggtgatcacccggctgaccaccgagtcgtggatgggtccggcctcgatggcgatggtcgccgcagcccagccctatctggettggttgacctacaccgccgaagccgctgcgcatgccggctcgcaggccatggcgteggcggccgcctacgaggcggcctatgcgatgacagtgccgccggaggtggtcgcggccaaccgggcgctgctggcggccctggtcgcgacgaacgtectggggatcaacacaccggcaatcatggcgaccgaagccctctatgccgagatgtgggctcaggacgctctggctatgtacggctacgcggccgcttcgggagccgccgggatgctgcaaccgttaagcccgccgtcgcagaccaccaacccgggcgggctggccgcccagtccgccgcggtcggctcggctgccgccaccgccgccgtcaaccaggtgagcgtagcggacctgatcagtagcctgcccaacgcggtgagtgggctcgcctccccagtcacatcggttctcgactcgacggggctgagcggaatcattgccgacatcgacgccctgctcgcgaccccgttcgtggcaaacatcatcaacagcgcagtcaacaccgccgcttggtatgtcaacgccgccatccccaccgcgatattcctagcaaatgccctgaacagtggggcgccggtagcgategccgaaggcgccatcgaggctgccgagggtgccgccagtgcggccgccgcggggttggcggactcggtgacgccagcggggctcggcgcaagtttaggcgaggccaccctggtcggccggctgtcagtgccggcggcctggtctacggccgcaccggcgacaaccgccggcgccacagcgctcgaaggcageggctggaccgtcgccgccgaagaagccggcccagttaccgggatgatgccgggaatggcctcggccgccaagggcaccggtgcctatgccgggccgcggtacggattcaagcccactgtcatgcccaaacaggtcgtcgtgtga (SEQ ID NO: 12) atggattttggcgccctgcctcccgagatcaacagcgctaggatgtatgctggcgctggagccggacctatgatggccgctggagccgcctggaatggactggctgccgaactgggcacaacagccgcttcctacgagtccgtgatcaccagactcaccacagagtectggatgggacctgccagcatggctatggtcgccgctgctcaaccctacctggcctggctgacctatacagctgaagccgctgctcacgccggaagccaagctatggctagcgccgccgcttatgaggccgcttatgccatgaccgtgcctcccgaggtcgtggctgccaacagagctctcctggccgccctcgtggctaccaacgtgctgggaatcaacacccccgctattatggccaccgaggctctgtacgctgagatgtgggcccaggatgccctcgccatgtacggatacgccgctgcttccggagctgctggaatgctgcagcccctgtcccccccttcccagaccaccaaccccggaggactggctgctcaaagcgctgctgtgggatccgctgctgctaccgctgccgtcaatcaggtcagcgtcgccgacctcatctccagcctgcctaacgctgtgagcggactggcctcccctgtcacatccgtgctcgatagcaccggcctgtccggcatcatcgccgacattgatgctctcctcgccaccccctttgtcgccaacatcatcaattccgccgtgaacaccgctgcctggtacgtcaacgctgccattcccaccgccatcttcctcgccaacgccctgaactccggagctectgtcgccatcgctgagggcgctattgaggctgctgaaggagccgctagcgctgctgctgctggactggctgatagcgtcacccctgctggactcggagctagcctgggagaagccaccctggteggcagactgtccgtgcctgctgcttggagcaccgctgctectgctacaaccgctggagctaccgctctggagggatccggatggacagtggctgctgaggaagctggacccgtgaccggaatgatgcctggcatggccagcgctgctaagggaaccggcgcctatgccggacccagatacggattcaagcccaccgtcatgcccaagcaggtcgtcgtctaa (SEQ ID NO: 13) MDFGALPPEINSARMYAGAGAGPMMAAGAAWNGLAAELGTTAASYESVITRLTTESWMGPASMAMVAAAQPYLAWLTYTAEAAAHAGSQAMASAAAYEAAYAMTVPPEVVAANRALLAALVATNVLGINTPAIMATEALYAEMWAQDALAMYGYAAASGAAGMLQPLSPPSQTTNPGGLAAQSAAVGSAAATAAVNQVSVADLISSLPNAVSGLASPVTSVLDSTGLSGIIADIDALLATPFVANIINSAVNTAAWYVNAAIPTAIFLANALNSGAPVAIAEGAIEAAEGAASAAAAGLADSVTPAGLGASLGEATLVGRLSVPAAWSTAAPATTAGATALEGSGWTVAAEEAGPVTGMMPGMASAAKGTGAYAGPRYGFKPTVMPKQVVV (SEQ ID NO: 14)  PPE51atggatttcgcactgttaccaccggaagtcaactccgcccggatgtacaccggccctggggcaggatcgctgttggctgccgcgggeggctgggattcgctggccgccgagttggccaccacagccgaggcatatggatcggtgctgtccggactggccgccttgcattggcgtggaccggcageggaatcgatggcggtgacggccgctccctatatcggttggctgtacacgaccgccgaaaagacacagcaaacagcgatccaagccagggcggcagcgctggccttcgageaagcatacgcaatgaccctgccgccaccggtggtagcggccaaccggatacagctgctagcactgatcgcgacgaacttatcggccagaacactgeggcgatcgcggccaccgaggcacagtacgccgagatgtgggcccaggacgccgccgcgatgtacggttacgccaccgcctcagcggctgcggccctgctgacaccgttctccccgccgcggcagaccaccaacccggccggcctgaccgctcaggccgccgcggtcagccaggccaccgacccactgtcgctgctgattgagacggtgacccaagcgctgcaagcgctgacgattccgagatcatccctgaggacttcaccttccttgacgccatattcgctggatatgccacggtaggtgtgacgcaggatgtcgagtectttgttgccgggaccatcggggccgagagcaacctaggccttttgaacgtcggcgacgagaatcccgeggaggtgacaccgggcgactttgggatcggcgagttggtttccgcgaccagteccggcggtggggtgtctgcgtcgggtgccggcggtgcggcgagcgtcggcaacacggtgctcgcgagtgtcggccgggcaaactcgattgggcaactatcggtcccaccgagctgggccgcgccctcgacgcgccctgtctcggcattgtcgcccgccggcctgaccacactcccggggaccgacgtggccgagcacgggatgccaggtgtaccgggggtgccagtggcagcagggcgagcctccggcgtectacctcgatacggggtteggctcacggtgatggcccacccacccgcggcagggtaa (SEQ ID NO: 15) atggattttgcgctgctgccgccggaagtgaacagcgcgcgcatgtataccggcccgggcgcgggcagcctgctggcggcggcgggcggctgggatagcctggcggcggaactggcgaccaccgcggaagcgtatggcagcgtgctgagcggcctggcggcgctgcattggcgcggcccggcggcggaaagcatggcggtgaccgcggcgccgtatattggctggctgtataccaccgcggaaaaaacccagcagaccgcgattcaggcgcgcgcggcggcgctggcgtttgaacaggcgtatgcgatgaccctgccgccgccggtggtggcggcgaaccgcattcagctgctggcgctgattgcgaccaacttttttggccagaacaccgcggcgattgcggcgaccgaagcgcagtatgcggaaatgtgggcgcaggatgcggcggcgatgtatggctatgcgaccgcgagcgcggcggcggcgctgctgaccccgtttagcccgccgcgccagaccaccaacccggegggcctgaccgcgcaggcggcggcggtgagccaggcgaccgatccgctgagcctgctgattgaaaccgtgacccaggcgctgcaggcgctgaccattccgagctttattccggaagattttacctttctggatgcgatttttgcgggctatgcgaccgtgggcgtgacccaggatgtggaaagattgtggcgggcaccattggcgcggaaagcaacctgggcctgctgaacgtgggcgatgaaaacccggcggaagtgaccccgggcgattttggcattggcgaactggtgagcgcgaccagcccgggcggcggcgtgagcgcgagcggcgcgggcggcgcggcgagegtgggcaacaccgtgctggcgagcgtgggccgcgcgaacagcattggccagctgagcgtgccgccgagctgggcggcgccgagcacccgcccggtgagcgcgctgagcccggcgggcctgaccaccctgccgggcaccgatgtggcggaacatggcatgccgggcgtgccgggcgtgccggtggcggcgggccgcgcgagcggcgtgctgccgcgctatggcgtgcgcctgaccgtgatggcgcatccgccggcggcgggcgaattt (SEQ ID NO: 16) atggatttcgctctgctcccccccgaggtgaatagcgctaggatgtacacaggacccggagctggaagcctcctggctgctgctggaggatgggactccctggctgccgagctcgctacaaccgctgaggcttacggaagcgtgctctccggcctggctgctctccattggagaggccctgctgccgagtccatggctgtcacagccgctccctacattggatggctgtacaccaccgccgagaagacccagcaaaccgctattcaggccagagctgccgccctggccttcgaacaggcctacgctatgacactccccccccctgtcgtggctgccaataggatccagctcctggccctcatcgccaccaacttcttcggccaaaacaccgctgccatcgctgccaccgaagcccagtacgccgaaatgtgggcccaggatgccgctgctatgtacggctatgccacagctagcgctgccgctgctctgctcacacccttcagcccccccaggcaaacaaccaaccctgccggactgacagcccaagctgctgccgtcagccaagctaccgaccccctgagcctcctgatcgaaaccgtgacacaggccctgcaggccctgaccattcccagattatccccgaggacttcacctttctggacgctatcttcgctggctacgccaccgtgggcgtgacacaagacgtcgagtecttcgtcgccggcacaatcggagccgagtccaaccteggactcctcaacgtcggcgacgaaaatcccgccgaagtgacacctggagacttcggcattggagaactcgtcagcgccacatcccctggcggaggagtgagcgcttccggagctggaggagctgcttccgtgggcaataccgtgctggccagcgtgggaagggccaactccattggccagctcagcgtccccccttcctgggctgccccttccacaaggcctgtgtccgctctcagccctgctggactgaccacactccctggcacagacgtggctgagcatggcatgcccggagtgcctggagtccctgtggctgctggcagagcttccggagtcctccctaggtatggcgtgaggctgacagtgatggctcatccccccgctgccggataa (SEQ ID NO: 17) MDFALLPPEVNSARMYTGPGAGSLLAAAGGWDSLAAELATTAEAYGSVLSGLAALHWRGPAAESMAVTAAPYIGWLYTTAEKTQQTAIQARAAALAFEQAYAMTLPPPVVAANRIQLLALIATNFFGQNTAAIAATEAQYAEMWAQDAAAMYGYATASAAAALLTPFSPPRQTTNPAGLTAQAAAVSQATDPLSLLIETVTQALQALTIPSFIPEDFTFLDAIFAGYATVGVTQDVESFVAGTIGAESNLGLLNVGDENPAEVTPGDFGIGELVSATSPGGGVSASGAGGAASVGNTVLASVGRANSIGQLSVPPSWAAPSTRPVSALSPAGLTTLPGTDVAEHGMPGVPGVPVAAGRASGVLPRYGVRLTVMAHPPAAG (SEQ ID NO: 18)  Rv3615catgacggaaaacttgaccgtccagcccgagegtctcggtgtactggcgtcgcaccatgacaacgcggcggtcgatgcctcctcgggcgtcgaagctgccgctggcctaggcgaatctgtggcgateactcacggtccgtactgctcacagttcaacgacacgttaaatgtgtacttgactgcccacaatgccctgggctcgtecttgcatacggccggtgtcgatctcgccaaaagtatcgaattgcggcgaagatatatagcgaggccgacgaagcgtggcgcaaggctatcgacgggttgtttacctga (SEQ ID NO: 19) atgaccgagaacctgaccgtgcagcctgagaggctgggagtgctggccagccaccacgacaacgctgccgtggacgcttccageggagtggaggctgctgctggactgggagagagcgtggccatcacccacggaccctactgcagccagttcaacgacaccctgaacgtgtacctgacagcccacaacgccctgggaagcagcctgcatacagccggcgtggacctggctaagtecctgaggatcgccgccaagatctacagcgaggccgacgaggcctggaggaaagccatcgacggcctgttcacctaa (SEQ ID NO: 20) MTENLTVQPERLGVLASHHDNAAVDASSGVEAAAGLGESVAITHGPYCSQFNDTLNVYLTAHNALGSSLHTAGVDLAKSLRIAAKIYSEADEAWRKAIDGLFT (SEQ ID NO: 21) 

In some embodiments, the latent Mtb antigen is Rv1733c, Rv2626c, Rv3407,or Rv2628c. In some embodiments, the latent Mtb antigen is Rv1733c orRv2626c. Additional latent Mtb antigens are well known to the skilledartisan.

A nucleotide sequence encoding the wild type latent Mtb antigen Rv1733cis shown in Table 2 as SEQ ID NO:22 (mycobacterial sequence; not codonoptimized), SEQ ID NO:23 (E. coli optimized), and an amino acid sequenceof wild type Rv1733c is shown in Table 2 as SEQ ID NO:24. Thesesequences include two transmembrane regions of Rv1733c. A nucleotidesequence encoding Rv1733c, whereby both transmembrane regions aredeleted is shown in Table 2 as SEQ ID NO:25 (E. coli optimized) and SEQID NO:26 (human optimized), and corresponding amino acid sequences areshown in Table 2 as SEQ ID NO:27 (E. coli optimized) and SEQ ID NO:28(human optimized) (Rv1733c_(ΔTM)). In some embodiments, only a portionof the first and/or second or both transmembrane regions are deleted. Inthe E. coli optimized nucleotide sequence (SEQ ID NO:23), an XmaIrestriction site was added, corresponding to an addition of amino acidsPG; and an XbaI restriction site was added, corresponding to an additionof amino acids SR (see underlined and bolded added sequences).

A nucleotide sequence encoding the latent Mtb antigen Rv2626c is shownin Table 2 as SEQ ID NO:29 (mycobacterial sequence; not codonoptimized), SEQ ID NO:30 (E. coli optimized), and SEQ ID NO:31 (humanoptimized), and an amino acid sequence of Rv2626c is shown in Table 2 asSEQ ID NO:32.

A nucleotide sequence encoding the latent Mtb antigen Rv3407 is shown inTable 2 as SEQ ID NO:33 (mycobacterial sequence; not codon optimized),SEQ ID NO:34 (E. coli optimized), and SEQ ID NO:35 (human optimized),and an amino acid sequence of Rv3407 is shown in Table 2 as SEQ IDNO:36.

A nucleotide sequence encoding the latent Mtb antigen Rv2628c is shownin Table 2 as SEQ ID NO:37 (mycobacterial sequence; not codon optimized)and as SEQ ID NO:38 (human optimized), and an amino acid sequence ofRv2628c is shown in Table 2 as SEQ ID NO:39.

TABLE 2  nucleotide sequence Construct amino acid sequence Rv1733catgatcgccacaacccgcgatcgtgaaggagccaccatgatcacgtttaggctgcgcttgccgtgccggacgatactgcgggtgttcagccgcaatccgctggtgcgtgggacggatcgactcgaggcggtcgtcatgctgctggccgtcacggtctcgctgctgactatcccgttcgccgccgcggccggcaccgcagtccaggattcccgcagccacgtctatgcccaccaggcccagacccgccatcccgcaaccgcgaccgtgatcgatcacgagggggtgatcgacagcaacacgaccgccacgtcagcgccgccgcgcacgaagatcaccgtgcctgcccgatgggtcgtgaacggaatagaacgcagcggtgaggtcaacgcgaagccgggaaccaaatccggtgaccgcgtcggcatttgggtcgacagtgccggtcagaggtcgatgaaccagaccgccggcccgtgccattgcggatgcggccctggccgccttgggactctggttgagcgtcgccgcggttgcgggcgccctgctggcgctcactcgggcgattctgatccgcgttcgcaacgccagttggcaacacgacatcgacagcctgttctgcacgcagcggtga (SEQ ID NO: 22)atgattgcgactacccgtgatcgtgagggcgcgaccatgatcacgttccgtctgcgtctgccgtgtcgcaccattttgcgcgtgttttcgcgtaacccgctggtccgcggtaccgaccgtctggaggccgttgtcatgctgctggcggttaccgtgagcctgctgacgatcccattcgcagcggcagaggcacggccgtccaagacagccgtagccatgtgtatgctcaccaggctcaaacccgtcacccggctactgccactgttatcgatcacgaaggcgtgattgactccaataccacggcaacctccgcaccgcctcgcaccaagattacggttcctgcgcgttgggtggtgaatggtattgaacgcagcggcgaagttaatgccaaaccgggtaccaaaagcggtgaccgtgtgggcatctgggtcgatagcgccggtcagaggtcgacgagccggcaccgccagcgcgtgcgatcgccgatgcggcgctggctgccctgggtctgtggctgagcgtggcagcggtcgccggtgcgttgctggcgctgacgcgcgcaattctgatccgcgttcgcaatgcgagctggcagcacgatattgatagcctgttttgcacccaacgt (SEQ ID NO: 23)MIATTRDREGATMITFRLRLPCRTILRVFSRNPLVRGTDRLEAVVMLLAVTVSLLTIPFAAAAGTAVQDSRSHVYAHQAQTRHPATATVIDHEGVIDSNTTATSAPPRTKITVPARWVVNGIERSGEVNAKPGTKSGDRVGIWVDSAGQLVDEPAPPARAIADAALAALGLWLSVAAVAGALLALTRAILIRVRNASWQHDIDSLFCTQR (SEQ ID NO: 24) Rv1733c_(ΔTM)atgattgcgactacccgtgatcgtgagggcgcgaccatgatcacgttccgtctgcgtctgccgtgtcgcaccattttgcgcgtgttttcgcgtaacccgctggtccgcggtaccgaccgtctggaggcc cccggggtccaagacagccgtagccatgtgtatgctcaccaggctcaaacccgtcacccggctactgccactgttatcgatcacgaaggcgtgattgactccaataccacggcaacctccgcaccgcctcgcaccaagattacggttcctgcgcgttgggtggtgaatggtattgaacgcagcggcgaagttaatgccaaaccgggtaccaaaagcggtgaccgtgtgggcatctgggtcgatagcgccggtcagctggtcgacgagccggcaccgccagcgcgtgcgatcgccgattctagacgcgcaattctgatccgcgttcgcaatgcgagctggcagcacgatattgatagcctgttttgcacccaacgt (SEQ ID NO: 25)atcgccaccaccagggacagggaaggcgctaccatgatcaccttcaggctgaggctcccctgcaggaccatcctgagggtgttcagcaggaaccccctggtgaggggcaccgacagactggaagccgtgcaggacagcaggagccacgtgtatgcccaccaggctcagaccaggcaccctgctaccgccaccgtgatcgaccacgagggcgtgatcgactccaacaccaccgccaccagcgctcctcccagaaccaagatcacagtgcccgccaggtgggtggtgaacggcatcgagaggagcggcgaggtgaacgccaagcctggaaccaagagcggcgacagggtgggcatttgggtcgatagcgccggccagctggtggatgaacctgctccccctgccagagccatcgccgatagggccatcctgatcagggtgaggaacgccagctggcagcacgacatcgacagcctgttctgcacccaaagg (SEQ ID NO: 26) MIATTRDREGATMITFRLRLPCRTILRVFSRNPLVRGTDRLEAPGVQDSRSHVYAHQAQTRHPATATVIDHEGVIDSNTTATSAPPRTKITVPARWVVNGIERSGEVNAKPGTKSGDRVGIWVDSAGQLVDEPAPPARAIADSRRAILIRVRNASWQHDIDSLFCTQR (SEQ ID NO: 27)IATTRDREGATMITFRLRLPCRTILRVFSRNPLVRGTDRLEAVQDSRSHVYAHQAQTRHPATATVIDHEGVIDSNTTATSAPPRTKITVPARWVVNGIERSGEVNAKPGTKSGDRVGIWVDSAGQLVDEPAPPARAIADRAILIRVRNASWQHDIDSLFCTQR (SEQ ID NO: 28) Rv2626catgaccaccgcacgcgacatcatgaacgcaggtgtgacctgtgttggcgaacacgagacgctaaccgctg ccgctcaatacatgcgtgagcacgacatcggcgcgttgccgatctgcggggacgacgaccggctgcacggcatgctcaccgaccgcgacattgtgatcaaaggcctggctgcgggcctagacccgaataccgccacggctggcgagttggcccgggacagcatctactacgtcgatgcgaacgcaagcatccaggagatgctcaacgtcatggaagaacatcaggtccgccgtgttccggtcatctcagagcaccgcttggtcggaatcgtcaccgaagccgacatcgcccgacacctgcccgagcacgccattgtgcagttcgtcaaggcaatctgctcgcccatggccctcgccagctag (SEQ ID NO: 29)atgaccacggcgcgtgatatcatgaatgcgggtgtcacctgtgttggcgagcacgaaacgttgaccgcagcagcacagtacatgcgcgaacatgatatcggcgcattgccgatttgcggcgacgatgatcgtctgcacggtatgctgaccgaccgcgatatcgttatcaagggtctggccgcaggcttggacccgaacaccgcgaccgccggt gaactggcacgtgacagcatctattacgtcgacgcgaacgccagcattcaagagatgctgaacgtgatgga agagcatcaggtgcgtcgtgtcccggttatcagcgaacatcgtctggttggtatcgttaccgaagccgacatc gcacgtcacctgccggagcacgcgattgttcagttcgtgaaagcgatttgcagcccgatggcgttggcgtc(SEQ ID NO: 30)acaacagccagggacatcatgaacgccggcgtgacctgcgtgggagagcatgaaaccctcaccgccgcc gcccaatacatgagggagcacgacatcggcgccctgcccatctgtggagacgacgacaggctgcacggc atgctgaccgacagggacatcgtgatcaagggcctggctgccggcctcgatcctaacaccgctacagccg gcgagctggccagagacagcatctactacgtggacgccaacgccagcatccaggagatgctcaacgtgat ggaggagcaccaggtgagaagggtgcctgtgatcagcgagcacaggctggtgggcatcgtgaccgagg ccgatatcgctaggcacctgcccgagcacgccatcgtgcagttcgtgaaggccatctgcagccccatggct ctggccagc (SEQ ID NO: 31) MTTARDIMAGVTCVGEHETLTAAAQYMREHDIGALPICGDDDRL HGMLTDRDIVIKGLAAGLDPNTATAGELARDSIYYVDANASIQEMLNVMEEHQVRRVPVISEHRLVGIVTEADIARHLPEHAIVQFVKAICSP MALAS (SEQ ID NO: 32)Rv3407atgcgtgctaccgttgggcttgtggaggcaatcggaatccgagaactaagacagcacgcatcgcgatacctcgcccgggttgaagccggcgaggaacttggcgtcaccaacaaaggaagacttgtggcccgactcatcccggtgcaggccgcggagcgttctcgcgaagccctgattgaatcaggtgtcctgattccggctcgtcgtccacaaaaccttctcgacgtcaccgccgaaccggcgcgcggccgcaagcgcaccctgtccgatgttctcaacgaaatgcgcgacgagcagtga (SEQ ID NO: 33)atgcgtgcgactgtgggtctggttgaggcgattggcattcgcgagctgcgccaacatgccagccgttacttggctcgtgtcgaggcgggtgaagaactgggcgtgacgaataagggtcgtctggtcgcccgtctgattccggttcaggcagctgagcgttctcgcgaggcgctgattgaatccggcgtcctgatcccggctcgccgtccgcaaaacctgctggacgtgacggcggagccagctcgtggtcgcaaacgcacgctgtctgatgtcctgaacgaaatgcgcgacgagcag (SEQ ID NO: 34)atgagggcgaccgtcgggctggtggaggcgataggtatccgggagttgcgacagcacgcatcacgatatctggcacgggtggaagctggggaggaactgggcgtgaccaacaaggggcggctggtcgcgaggctgatccccgtgcaggccgccgagcggtcccgcgaagccctcatcgagtctggggtgctcattccagcacgcaggccgcaaaatctcctggacgtcactgcggagcccgccagaggcagaaagaggacgctgagtgacgtgctgaacgagatgagggacgaacag (SEQ ID NO: 35)MRATVGLVEAIGIRELRQHASRYLARVEAGEELGVTNKGRLVARLIPVQAAERSREALIESGVLIPARRPQNLLDVTAEPARGRKRTLSDVLN EMRDEQ (SEQ ID NO: 36)Rv2628atgtccacgcaacgaccgaggcactccggtattcgggctgttggcccctacgcatgggccggccgatgtggtcggataggcaggtggggggtgcaccaggaggcgatgatgaatctagcgatatggcacccgcgcaaggtgcaatccgccaccatctatcaggtgaccgatcgctcgcacgacgggcgcacagcacgggtgcctggtgacgagatcactagcaccgtgtccggttggttgtcggagttgggcacccaaagcccgttggccgatgagcttgcgcgtgcggtgcggatcggcgactggcccgctgcgtacgcaatcggtgagcacctgtccgttgagattgccgttgcggtctaa (SEQ ID NO: 37)atgagcacccagagacccaggcacagcggcattagggccgtgggaccttatgatgggccggcagatgcggaaggatcggcagatggggcgtgcaccaagaggccatgatgaacctggccatctggcaccccaggaaggtgcagagcgccaccatctaccaggtgaccgacaggagccatgacggaaggaccgccagagtgcccggcgatgagatcaccagcaccgtgagcggctggctgagcgaactgggcacccaatcccccctggctgatgaactggccagggctgtgaggatcggcgattggcctgccgcctatgccatcggcgagcatctgagcgtggagatcgccgtggccgtgtaa (SEQ ID NO: 38)MSTQRPRHSGIRAVGPYAWAGRCGRIGRWGVHQEAMMNLAIWHPRKVQSATIYQVTDRSHDGRTARVPGDEITSTVSGWLSELGTQSPLADELARAVRIGDWPAAYAIGEHLSVEIAVAV (SEQ ID NO: 39)

In some embodiments, the resuscitation Mtb antigen is RpfB, RpfD, orRpfE. In some embodiments, the resuscitation Mtb antigen is RpfB orRpfD. Additional resuscitation antigens include, but are not limited to,Rv0867c, Rv0288, Rv1009, Rv0685, Rv0824c, Rv2744c, Rv3347c, Rv1130,Rv1169c, Rv1884c, Rv2389c, and Rv2450c. In some embodiments, theresuscitation antigen is Rv0867c, Rv1884c, or Rv2389c. Additionalresuscitation Mtb antigens are well known to the skilled artisan. Insome embodiments, the resuscitation antigen is not any one or more ofthe following: Rv0867c, Rv0288, Rv1009, Rv0685, Rv0824c, Rv2744c,Rv3347c, Rv1130, Rv1169c, Rv1884c, Rv2389c, and Rv2450c. In someembodiments, the resuscitation antigen is not Rv0867c, Rv1884c, orRv2389c.

The resuscitation Mtb antigen RpfB is also known as Rv1009. A nucleotidesequence encoding the resuscitation Mtb antigen RpfB is shown in Table 3as SEQ ID NO:40 (mycobacterial sequence; not codon optimized) and SEQ IDNO:41 (mycobacterial sequence; signal sequence deleted), and amino acidsequences of RpfB corresponding thereto are shown in Table 3 as SEQ IDNO:42 and SEQ ID NO:43, respectively.

The resuscitation Mtb antigen RpfD is also known as Rv2389c. Anucleotide sequence encoding the resuscitation Mtb antigen RpfD is shownin Table 3 as SEQ ID NO:44 (mycobacterial sequence; not codonoptimized), SEQ ID NO:45 (E. coli optimized; leader sequence deleted)and SEQ ID NO:46 (human optimized; leader sequence present), and aminoacid sequences of RpfD are shown in Table 3 as SEQ ID NO:47 (E. colioptimized) and SEQ ID NO:48 (mycobacterial sequence and humanoptimized).

The resuscitation Mtb antigen RpfE is also known as Rv2450c. Anucleotide sequence encoding the resuscitation Mtb antigen RpfE is shownin Table 3 as SEQ ID NO:49 (mycobacterial sequence; not codonoptimized), and an amino acid sequence of RpfE is shown in Table 3 asSEQ ID NO:50.

TABLE 3 nucleotide sequence Construct amino acid sequence RpfBatgttgcgcctggtagtcggtgcgctgctgctggtgttggcgttcgccggtggctatgcggtcgccgcatgcaaaacggtgacgttgaccgtcgacggaaccgcgatgcgggtgaccacgatgaaatcgcgggtgatcgacatcgtcgaagagaacgggttctcagtcgacgaccgcgacgacctgtatcccgcggccggcgtgcaggtccatgacgccgacaccatcgtgctgcggcgtagccgtccgctgcagatctcgctggatggtcacgacgctaagcaggtgtggacgaccgcgtcgacggtggacgaggcgctggcccaactcgcgatgaccgacacggcgccggccgcggcttctcgcgccagccgcgtcccgctgtccgggatggcgctaccggtcgtcagcgccaagacggtgcagctcaacgacggcgggttggtgcgcacggtgcacttgccggcccccaatgtcgcggggctgctgagtgcggccggcgtgccgctgttgcaaagcgaccacgtggtgcccgccgcgacggccccgatcgtcgaaggcatgcagatccaggtgacccgcaatcggatcaagaaggtcaccgagcggctgccgctgccgccgaacgcgcgtcgtgtcgaggacccggagatgaacatgagccgggaggtcgtcgaagacccgggggttccggggacccaggatgtgacgttcgcggtagctgaggtcaacggcgtcgagaccggccgtttgcccgtcgccaacgtcgtggtgaccccggcccacgaagccgtggtgcgggtgggcaccaagcccggtaccgaggtgcccccggtgatcgacggaagcatctgggacgcgatcgccggctgtgaggccggtggcaactgggcgatcaacaccggcaacgggtattacggtggtgtgcagtttgaccagggcacctgggaggccaacggcgggctgcggtatgcaccccgcgctgacctcgccacccgcgaagagcagatcgccgttgccgaggtgacccgactgcgtcaaggttggggcgcctggccggtatgtgctgcacgagcgggtgcgcgctga (SEQ ID NO: 40)gcatgcaaaacggtgacgttgaccgtcgacggaaccgcgatgcgggtgaccacgatgaaatcgcgggtgatcgacatcgtcgaagagaacgggttctcagtcgacgaccgcgacgacctgtatcccgcggccggcgtgcaggtccatgacgccgacaccatcgtgctgcggcgtagccgtccgctgcagatctcgctggatggtcacgacgctaagcaggtgtggacgaccgcgtcgacggtggacgaggcgctggcccaactcgcgatgaccgacacggcgccggccgcggcttctcgcgccagccgcgtcccgctgtccgggatggcgctaccggtcgtcagcgccaagacggtgcagctcaacgacggcgggttggtgcgcacggtgcacttgccggcccccaatgtcgcggggctgctgagtgcggccggcgtgccgctgttgcaaagcgaccacgtggtgcccgccgcgacggccccgatcgtcgaaggcatgcagatccaggtgacccgcaatcggatcaagaaggtcaccgagcggctgccgctgccgccgaacgcgcgtcgtgtcgaggacccggagatgaacatgagccgggaggtcgtcgaagacccgggggttccggggacccaggatgtgacgttcgcggtagctgaggtcaacggcgtcgagaccggccgtttgcccgtcgccaacgtcgtggtgaccccggcccacgaagccgtggtgcgggtgggcaccaagcccggtaccgaggtgcccccggtgatcgacggaagcatctgggacgcgatcgccggctgtgaggccggtggcaactgggcgatcaacaccggcaacgggtattacggtggtgtgcagtttgaccagggcacctgggaggccaacggcgggctgcggtatgcaccccgcgctgacctcgccacccgcgaagagcagatcgccgttgccgaggtgacccgactgcgtcaaggttggggcgcctggccggtatgtgctgcacgagcgggtgcgcgctga (SEQ ID NO: 41)MLRLVVGALLLVLAFAGGYAVAACKTVTLTVDGTAMRVTTMKSRVIDIVEENGFSVDDRDDLYPAAGVQVHDADTIVLRRSRPLQISLDGHDAKQVWTTASTVDEALAQLAMTDTAPAAASRASRVPLSGN4ALPVVSAKTVQLNDGGLVRTVHLPAPNVAGLLSAAGVPLLQSDHVVPAATAPIVEGMQ1QVTRNR1KKVTERLPLPPNARRVEDPEMNMSREVVEDPGVPGTQDVTFAVAEVNGVETGRLPVANVVVTPAHEAVVRVGTKPGTEVPPVTDGSIWDAIAGCEAGGNWATNTGNGYYGGVQFDQGTWEANGGLRYAPRADLATREEQIAVAEVTRLRQGWGAWPVCAARAGAR (SEQ ID NO: 42)ACKTVTLTVDGTAMRVTTMKSRVTDIVEENGFSVDDRDDLYPAAGVQVHDADTIVLRRSRPLQISLDGHDAKQVWTTASTVDEALAQLAMTDTAPAAASRASRVPLSGMALPVVSAKTVQLNDGGLVRTVHLPAPNVAGLLSAAGVPLLQSDHVVPAATAPrVEGMQIQVTRNRIKKVTERLPLPPNARRVEDPEMNMSREVVEDPGVPGTQDVTFAVAEVNGVETGRLPVANVVVTPAHEAVVRVGTKPGTEVPPVIDGSIWDAIAGCEAGGNWAINTGNGYYGGVQFDQGTWEANGGLRYAPRADLATREEQIAVAEVTRLRQGWGAWPVCAARAGAR (SEQ ID NO: 43) RpfDatgacaccgggtttgcttactactgcgggtgctggccgaccacgtgacaggtgcgccaggatcgtatgcacggtgttcatcgaaaccgccgttgtcgcgaccatgtttgtcgcgttgttgggtctgtccaccatcagctcgaaagccgacgacatcgattgggacgccatcgcgcaatgcgaatccggcggcaattgggcggccaacaccggtaacgggttatacggtggtctgcagatcagccaggcgacgtgggattccaacggtggtgtcgggtcgccggcggccgcgagtccccagcaacagatcgaggtcgcagacaacattatgaaaacccaaggcccgggtgcgtggccgaaatgtagttcttgtagtcagggagacgcaccgctgggctcgctcacccacatcctgacgttcctcgcggccgagactggaggttgttcggggagcagggacgattga (SEQ ID NO: 44)aagcttttgctgggcctgagcaccattagcagcaaagcggatgacatcgactgggatgcgattgcgcagtgtgagagcggtggcaattgggcagcgaataccggcaatggcctgtacggcggtctgcagatctcccaggcgacgtgggacagcaatggtggcgtcggcagcccggctgccgcgtccccacaacaacagatcgaggtggcagataacattatgaaaacgcagggtccgggtgcttggccaaaatgctccagctgcagccagggtgacgcaccgctgggcagcctgacccacattctgacgttcctggcagcggaaaccggtggttgtagcggtagccgcgatgac (SEQ ID NO: 45)acccccggactcctcaccacagctggagctggcaggcccagagacagatgcgccaggatcgtgtgcaccgtgttcatcgagaccgccgtggtggctaccatgttcgtggccctgctgggcctgagcaccatcagcagcaaggccgacgacatcgactgggacgccatcgcccagtgtgaatccggcggaaactgggccgccaataccggcaatggcctgtacggcggcctgcagatcagccaggctacctgggactccaacggaggagtgggaagccctgccgctgatcccctcagcagcagatcgaggtggccgacaacatcatgaagacccaaggccctggcgcctggcctaagtgttccagctgtagccagggcgatgctcctctgggcagcctgacccacatcctgacctttctcgccgccgagacaggcggatgtagcggaagcagggacgactaatga (SEQ ID NO: 46) LLGLSTISSKADDIDWDAIAQCESGGNWAANTGNGLYGGLQISQATWDSNGGVGSPAAASPQQQIEVADNIMKTQGPGAWPKCSSCSQGDAPLGSLTHILTFLAAETGGCSGSRDD (SEQ ID NO: 47)TPGLLTTAGAGRPRDRCARIVCTVFIETAVVATMFVALLGLSTISSKADDIDWDAIAQCESGGNWAANTGNGLYGGLQISQATWDSNGGVGSPAAASPQQQIEVADNIMKTQGPGAWPKCSSCSQGDAPLGSLTHILTFLAAETGGCSGSRDD (SEQ ID NO: 48) RpfEttgaagaacgcccgtacgacgctcatcgccgccgcgattgccgggacgttggtgaccacgtcaccagccggtatcgccaatgccgacgacgcgggcttggacccaaacgccgcagccggcccggatgccgtgggctttgacccgaacctgccgccggccccggacgctgcacccgtcgatactccgccggctccggaggacgcgggctttgatcccaacctccccccgccgctggccccggacttcctgtccccgcctgcggaggaagcgcctcccgtgcccgtggcctacagcgtgaactgggacgcgatcgcgcagtgcgagtccggtggaaactggtcgatcaacaccggtaacggttactacggcggcctgcggttcaccgccggcacctggcgtgccaacggtggctcggggtccgcggccaacgcgagccgggaggagcagatccgggtggctgagaacgtgctgcgttcgcagggtatccgcgcctggccggtctgcggccgccgcggctga (SEQ ID NO: 49) LKNARTTLIAAAIAGTLVTTSPAGIANADDAGLDPNAAAGPDAVGFDPNLPPAPDAAPVDTPPAPEDAGFDPNLPPPLAPDFLSPPAEEAPPVPVAYSVNWDAIAQCESGGNWSINTGNGYYGGLRFTAGTWRANGGSGSAANASREEQIRVAENVLRSQGIRAWPVCGRRG (SEQ ID NO: 50)

In some embodiments, the fusion protein comprises at least fourMycobacterium tuberculosis (Mtb) antigens. In some embodiments, thefusion protein comprises at least five Mtb antigens. In someembodiments, the fusion protein comprises at least six Mtb antigens. Insome embodiments, the fusion protein comprises from at least three to atleast six Mtb antigens. In some embodiments, the fusion proteincomprises from at least three to at least five Mtb antigens. In someembodiments, the fusion protein comprises at least three or at leastfour Mtb antigens. In some embodiments, the fusion protein comprisesfrom at least four to at least six Mtb antigens. In some embodiments,the fusion protein comprises at least four or at least five Mtbantigens.

In some embodiments, the fusion protein comprises ESAT6, Rv1733c,Rv2626c, and RpfD Mtb antigens. In some embodiments, the fusion proteincomprises ESAT6, Rv1733c, Rv2626c, and RpfB Mtb antigens. In someembodiments, the fusion protein comprises RpfB, ESAT6, Rv1733c, andRv2626c Mtb antigens. In some embodiments, the fusion protein comprisesAg85B, ESAT6, Rv1733c, Rv2626c, and RpfD Mtb antigens. In someembodiments, the fusion protein comprises Ag85B, ESAT6, Rv1733c,Rv2626c, and RpfB Mtb antigens. In some embodiments, the fusion proteincomprises PPE51, Rv1733c, Rv2628c, and RpfD Mtb antigens. In someembodiments, the fusion protein comprises PPE51, Rv1733c, Rv2628c, andRpfB Mtb antigens. In some embodiments, the fusion protein comprisesRv3407, Rv1733c, Rv2626c, and RpfB Mtb antigens. In some embodiments,the fusion protein comprises Rv3407, Rv1733c, Rv2626c, and RpfD Mtbantigens.

In any of the embodiments of fusion proteins set forth herein, theindividual Mtb antigens can be present in any order. For example, for afusion protein comprising ESAT6, Rv1733c, Rv2626c, and RpfD Mtbantigens, the first (or N-terminal) antigen may be ESAT6, Rv1733c,Rv2626c, or RpfD; the second antigen may be ESAT6, Rv1733c, Rv2626c, orRpfD; the third antigen may be ESAT6, Rv1733c, Rv2626c, or RpfD; and thefourth (or C-terminal) antigen may be ESAT6, Rv1733c, Rv2626c, or RpfD.Likewise for every fusion protein disclosed herein.

Individual Mtb antigens may be linked together in a C-terminus toN-terminus manner without any linker (i.e., the C-terminus of ESAT6linked directly to the N-terminus of Rv1733c). Alternately, a linker maybe present between any two Mtb antigens within any of the fusionproteins disclosed herein. In some embodiments, the linker is a segmentof DNA or RNA optionally containing one or more restrictions sites,wherein the linker is inserted between nucleic acid molecules encodingtwo Mtb antigens of any of the fusion proteins disclosed herein. Table 5shows representative primers for particular Mtb antigens used tointroduce restriction sites into a fusion protein construct.

In some embodiments, the fusion protein comprisesESAT6-Rv1733c-Rv2626c-RpfD (Construct A; nucleotide sequence is SEQ IDNO:51 (E. coli optimized; inserted EcoRI, SacI, and HindIII restrictionsites, respectively, are bolded and underlined) and SEQ ID NO:52 (humanoptimized; inserted BstBI, PvuI, and AscI restriction sites,respectively, are bolded and underlined); corresponding amino acidsequences are SEQ ID NO:53 (E. coli optimized) and SEQ ID NO:54 (humanoptimized); see Table 4).

In some embodiments, the fusion protein comprisesESAT6-Rv1733c-Rv2626c-RpfB (Construct B; nucleotide sequence is SEQ IDNO:55, wherein inserted EcoRI, SacI, and HindIII restrictions sites,respectively, are bolded and underlined; amino acid sequence is SEQ IDNO:56; see Table 4).

In some embodiments, the fusion protein comprisesRpfB-ESAT6-Rv1733c-Rv2626c (Construct C; nucleotide sequence is SEQ IDNO:57, wherein inserted BamHI, EcoRI, and SacI restrictions sites,respectively, are bolded and underlined; amino acid sequence is SEQ IDNO:58; see Table 4).

In some embodiments, the fusion protein comprisesAg85B-ESAT6-Rv1733c-Rv2626c-RpfD (Construct D; nucleotide sequence isSEQ ID NO:59 (E. coli optimized; inserted BamHI, EcoR1, SacI, andHindIII restriction sites, respectively, are bolded and underlined) andSEQ ID NO:60 (human optimized; inserted XmaI, BstBI, PvuI, and AscIrestriction sites, respectively, are bolded and underlined); amino acidsequence is SEQ ID NO:61 (E. coli optimized) and SEQ ID NO:62 (humanoptimized); see Table 4).

In some embodiments, the fusion protein comprisesPPE51-Rv1733c-Rv2628c-RpfD (Construct E; nucleotide sequence is SEQ IDNO:63, wherein inserted EcoRI, SacI, and HindIII restrictions sites,respectively, are bolded and underlined; amino acid sequence is SEQ IDNO:64; see Table 4).

In some embodiments, the fusion protein comprisesPPE51-Rv1733c-Rv2628c-RpfB (Construct F; nucleotide sequence is SEQ IDNO:65, wherein inserted EcoRI, SalI, and HindIII restrictions sites,respectively, are bolded and underlined; amino acid sequence is SEQ IDNO:66; see Table 4).

In some embodiments, the fusion protein comprisesRv3407-Rv1733c-Rv2626c-RpfB (Construct G; nucleotide sequence is SEQ IDNO:67, wherein inserted EcoRI, SacI, and HindIII restrictions sites,respectively, are bolded and underlined; amino acid sequence is SEQ IDNO:68; see Table 4).

In some embodiments, the fusion protein comprisesRv3407-Rv1733c-Rv2626c-RpfD (Construct H; nucleotide sequence is SEQ IDNO:69, wherein inserted EcoRI, SacI, and HindIII restrictions sites,respectively, are bolded and underlined; amino acid sequence is SEQ IDNO:70; see Table 4).

In some embodiments, the fusion protein comprisesPPE51-Rv1733c-Rv2626c-RpfD (Construct I; nucleotide sequence is SEQ IDNO:71, wherein inserted EcoRI, SacI, and HindIII restrictions sites,respectively, are bolded and underlined; amino acid sequence is SEQ IDNO:72; see Table 4).

In some embodiments, the fusion protein comprisesPPE51-Rv1733c-Rv2626c-RpfB (Construct J; nucleotide sequence is SEQ IDNO:73, wherein inserted EcoRI, SacI, and HindIII restrictions sites,respectively, are bolded and underlined; amino acid sequence is SEQ IDNO:74; see Table 4).

nucleotide sequence Construct amino acid sequence Aatgacagagcagcagtggaatttcgcgggtatcgaggccgcggcaagcgcaatccagggaaatgtcacgtccattcattccctccttgacgaggggaagcagtccctgaccaagctcgcagcggcctggggcggtagcggttcggaggcgtaccagggtgtccagcaaaaatgggacgccacggctaccgagctgaacaacgcgctgcagaacctggcgcggacgatcagcgaagccggtcaggcaatggcttcgaccgaaggcaacgtcactgggatgttcgca gaattc atgattgcgactacccgtgatcgtgagggcgcgaccatgatcacgttccgtctgcgtctgccgtgtcgcaccattttgcgcgtgttttcgcgtaacccgctggtccgcggtaccgaccgtctggaggcccccggggtccaagacagccgtagccatgtgtatgctcaccaggctcaaacccgtcacccggctactgccactgttatcgatcacgaaggcgtgattgactccaataccacggcaacctccgcaccgcctcgcaccaagattacggttcctgcgcgttgggtggtgaatggtattgaacgcagcggcgaagttaatgccaaaccgggtaccaaaagcggtgaccgtgtgggcatctgggtcgatagcgccggtcagctggtcgacgagccggcaccgccagcgcgtgcgatcgccgattctagacgcgcaattctgatccgcgttcgcaatgcgagctggcagcacgatattgatagcctgttttgcacccaa cgtgagctc atgaccacggcgcgtgatatcatgaatgcgggtgtcacctgtgttggcgagcacgaaacgttgaccgcagcagcacagtacatgcgcgaacatgatatcggcgcattgccgatttgcggcgacgatgatcgtctgcacggtatgctgaccgaccgcgatatcgttatcaagggtctggccgcaggcttggacccgaacaccgcgaccgccggtgaactggcacgtgacagcatctattacgtcgacgcgaacgccagcattcaagagatgctgaacgtgatggaagagcatcaggtgcgtcgtgtcccggttatcagcgaacatcgtctggttggtatcgttaccgaagccgacatcgcacgtcacctgccggagcacgcgattgttcagttcgtgaaagcgatttgcagcccgatggcgttggcgtct aagctt ttgctgggcctgagcaccattagcagcaaagcggatgacatcgactgggatgcgattgcgcagtgtgagagcggtggcaattgggcagcgaataccggcaatggcctgtacggcggtctgcagatctcccaggcgacgtgggacagcaatggtggcgtcggcagcccggctgccgcgtccccacaacaacagatcgaggtggcagataacattatgaaaacgcagggtccgggtgcttggccaaaatgctccagctgcagccagggtgacgcaccgctgggcagcctgacccacattctgacgttcctggcagcggaaaccggtggttgtagcggtagccgcgatga c =(SEQ ID NO: 51)atgaccgagcagcagtggaacttcgccggcatcgaagctgccgctagcgccatccaaggcaacgtgaccagcatccacagcctgctggacgagggcaagcagagcctgaccaagctggctgctgcttggggcggatccggaagcgaagcctaccagggcgtgcagcagaagtgggacgccacagccaccgagctgaacaacgccctgcagaacctcgccagaaccatcagcgaggccggacaggctatggccagcacagagggcaatgtgaccggcatgttcgcc ttcgaa atcgccaccaccagggacagggaaggcgctaccatgatcaccttcaggctgaggctcccctgcaggaccatcctgagggtgttcagcaggaaccccctggtgaggggcaccgacagactggaagccgtgcaggacagcaggagccacgtgtatgcccaccaggctcagaccaggcaccctgctaccgccaccgtgatcgaccacgagggcgtgatcgactccaacaccaccgccaccagcgctcctcccagaaccaagatcacagtgcccgccaggtgggtggtgaacggcatcgagaggagcggcgaggtgaacgccaagcctggaaccaagagcggcgacagggtgggcatttgggtcgatagcgccggccagctggtggatgaacctgctccccctgccagagccatcgccgatagggccatcctgatcagggtgaggaacgccagctggcagcacgacatcgacagcctgttctgcacccaaagg cgatcg acaacagccagggacatcatgaacgccggcgtgacctgcgtgggagagcatgaaaccctcaccgccgccgcccaatacatgagggagcacgacatcggcgccctgcccatctgtggagacgacgacaggctgcacggcatgctgaccgacagggacatcgtgatcaagggcctggctgccggcctcgatcctaacaccgctacagccggcgagctggccagagacagcatctactacgtggacgccaacgccagcatccaggagatgctcaacgtgatggaggagcaccaggtgagaagggtgcctgtgatcagcgagcacaggctggtgggcatcgtgaccgaggccgatatcgctaggcacctgcccgagcacgccatcgtgcagttcgtgaaggccatctgcagccccatggctctggccagc ggcgcgcc cacccccggactcctcaccacagctggagctggcaggcccagagacagatgcgccaggatcgtgtgcaccgtgttcatcgagaccgccgtggtggctaccatgttcgtggccctgctgggcctgagcaccatcagcagcaaggccgacgacatcgactgggacgccatcgcccagtgtgaatccggcggaaactgggccgccaataccggcaatggcctgtacggcggcctgcagatcagccaggctacctgggactccaacggaggagtgggaagccctgccgctgcttcccctcagcagcagatcgaggtggccgacaacatcatgaagacccaaggccctggcgcctggcctaagtgttccagctgtagccagggcgatgctcctctgggcagcctgacccacatcctgacctttctcgccgccgagacaggcggatgtagcggaagcagggacgactaatgatag (SEQ ID NO: 52)MTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWDATATELNNALQNLARTISEAGQAMASTEGNVTGMFAEFMIATTRDREGATMITFRLRLPCRTILRVFSRNPLVRGTDRLEAPGVQDSRSHVYAHQAQTRHPATATVIDHEGVIDSNTTATSAPPRTKITVPARWVVNGIERSGEVNAKPGTKSGDRVGIWVDSAGQLVDEPAPPARAIADSRRAILIRVRNASWQHDIDSLFCTQRELMTTARDIMNAGVTCVGEHETLTAAAQYMREHDIGALPICGDDDRLHGMLTDRDIVIKGLAAGLDPNTATAGELARDSIYYVDANASIQEMLNVMEEHQVRRVPVISEHRLVGIVTEADIARHLPEHAIVQFVKAICSPMALASKLLLGLSTISSKADDIDWDAIAQCESGGNWAANTGNGLYGGLQISQATWDSNGGVGSPAAASPQQQIEVADNIMKTQGPGAWPKCSSCSQGDAPLGSLTHILTFLAAETGGCSGSRDD (SEQ ID NO: 53)MTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWDATATELNNALNLARTISEAGQAMASTEGNVTGMFAFEIATTRDREGATMITFRLRLPCRTILRVFSRNPLVRGTDRLEAVQDSRSHVYAHQAQTRHPATATVIDHEGVIDSNTTATSAPPRTKITVPARWVVNGIERSGEVNAKPGTKSGDRVGIWVDSAGQLVDEPAPPARAIADRAILIRVRNASWQHDIDSLFCTQRRSTTARDIMNAGVTCVGEHETLTAAAQYMREHDIGALPICGDDDRLHGMLTDRDIVIKGLAAGLDPNTATAGELARDSIYYVDANASIQEMLNVMEEHQVRRVPVISEHRLVGIVTEADIARHLPEHAIVQFVKAICSPMALASGAPTPGLLTTAGAGRPRDRCARIVCTVFIETAVVATMFVALLGLSTISSKADDIDWDAIAQCESGGNWAANTGNGLYGGLQISQATWDSNGGVGSPAAASPQQQIEVADNIMKTQGPGAWPKCSSCSQGDAPLGSLTHILTFLAAETGGCSGSRDD (SEQ ID NO: 54) Batgacagagcagcagtggaatttcgcgggtatcgaggccgcggcaagcgcaatccagggaaatgtcacgtccattcattccctccttgacgaggggaagcagtccctgaccaagctcgcagcggcctggggcggtagcggttcggaggcgtaccagggtgtccagcaaaaatgggacgccacggctaccgagctgaacaacgcgctgcagaacctggcgcggacgatcagcgaagccggtcaggcaatggatcgaccgaaggcaacgtcactgggatgttcgca gaattc atgattgcgactacccgtgatcgtgagggcgcgaccatgatcacgttccgtctgcgtctgccgtgtcgcaccattttgcgcgtgttttcgcgtaacccgctggtccgcggtaccgaccgtctggaggccgttgtcatgctgctggcggttaccgtgagcctgctgacgatcccattcgcagcggcagctggcacggccgtccaagacagccgtagccatgtgtatgctcaccaggctcaaacccgtcacccggctactgccactgttatcgatcacgaaggcgtgattgactccaataccacggcaacctccgcaccgcctcgcaccaagattacggttcctgcgcgttgggtggtgaatggtattgaacgcagcggcgaagttaatgccaaaccgggtaccaaaagcggtgaccgtgtgggcatctgggtcgatagcgccggtcagctggtcgacgagccggcaccgccagcgcgtgcgatcgccgatgcggcgctggctgccctgggtctgtggctgagcgtggcagcggtcgccggtgcgttgctggcgctgacgcgcgcaattctgatccgcgttcgcaatgcgagctggcagcacgatattgatagcctgttttgcacccaacgt gagc tcatgaccacggcgcgtgatatcatgaatgcgggtgtcacctgtgttggcgagcacgaaacgttgaccgcagcagcacagtacatgcgcgaacatgatatcggcgcattgccgatttgcggcgacgatgatcgtctgcacggtatgctgaccgaccgcgatatcgttatcaagggtctggccgcaggcttggacccgaacaccgcgaccgccggtgaactggcacgtgacagcatctattacgtcgacgcgaacgccagcattcaagagatgctgaacgtgatggaagagcatcaggtgcgtcgtgtcccggttatcagcgaacatcgtctggttggtatcgttaccgaagccgacatcgcacgtcacctgccggagcacgcgattgttcagttcgtgaaagcgatttgcagcccgatggcgttggcgtctcgtcaaaagggcgacacaaaatttattctaa atgcaaagctt gcatgcaaaacggtgacgttgaccgtcgacggaaccgcgatgcgggtgaccacgatgaaatcgcgggtgatcgacatcgtcgaagagaacgggttctcagtcgacgaccgcgacgacctgtatcccgcggccggcgtgcaggtccatgacgccgacaccatcgtgctgcggcgtagccgtccgctgcagatctcgctggatggtcacgacgctaagcaggtgtggacgaccgcgtcgacggtggacgaggcgctggcccaactcgcgatgaccgacacggcgccggccgcggcttctcgcgccagccgcgtcccgctgtccgggatggcgctaccggtcgtcagcgccaagacggtgcagctcaacgacggcgggttggtgcgcacggtgcacttgccggcccccaatgtcgcggggctgctgagtgcggccggcgtgccgctgttgcaaagcgaccacgtggtgcccgccgcgacggccccgatcgtcgaaggcatgcagatccaggtgacccgcaatcggatcaagaaggtcaccgagcggctgccgctgccgccgaacgcgcgtcgtgtcgaggacccggagatgaacatgagccgggaggtcgtcgaagacccgggggttccggggacccaggatgtgacgttcgcggtagctgaggtcaacggcgtcgagaccggccgtttgcccgtcgccaacgtcgtggtgaccccggcccacgaagccgtggtgcgggtgggcaccaagcccggtaccgaggtgcccccggtgatcgacggaagcatctgggacgcgatcgccggctgtgaggccggtggcaactgggcgatcaacaccggcaacgggtattacggtggtgtgcagtttgaccagggcacctgggaggccaacggcgggctgcggtatgcaccccgcgctgacctcgccacccgcgaagagcagatcgccgttgccgaggtgacccgactgcgtcaaggttggggcgcctggccggtatgtgctgcacgagcgggtgcgcgctga (SEQ ID NO: 55)MTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWDATATELNNALQNLARTISEAGQAMASTEGNVTGMFAEFMIATTRDREGATMITFRLRLPCRTILRVFSRNPLVRGTDRLEAVVMLLAVTVSLLTIPFAAAAGTAVQDSRSHVYAHQAQTRHPATATVIDHEGVIDSNTTATSAPPRTKITVPARWVVNGIERSGEVNAKPGTKSGDRVGIWVDSAGQLVDEPAPPARAIADAALAALGLWLSVAAVAGALLALTRAILIRVRNASWQHDIDSLFCTQRELMTTARDIMNAGVTCVGEHETLTAAAQYMREHDIGALPICGDDDRLHGMLTDRDTVIKGLAAGLDPNTATAGELARDSIYYVDANASIQEMLNVMEEHQVRRVPVISEHRLVGIVTEADIARHLPEHAIVQFVKAICSPMALASRQKGDTKFILNAKLACKTVTLTVDGTAMRVTTMKSRVIDIVEENGFSVDDRDDLYPAAGVQVHDADTIVLRRSRPLQISLDGHDAKQVWTTASTVDEALAQLAMTDTAPAAASRASRVPLSGMALPVVSAKTVQLNDGGLVRTVHLPAPNVAGLLSAAGVPLLQSDHVVPAATAPIVEGMQIQVTRNRIKKVTERLPLPPNARRVEDPEMNMSREVVEDPGVPGTQDVTFAVAEVNGVETGRLPVANVVVTPAHEAVVRVGTKPGTEVPPVIDGSIWDAIAGCEAGGNWAINTGNGYYGGVQFDQGTWEANGGLRYAPRADLATREEQIAVAEVTRLRQGWGAWPVCAARAGAR (SEQ ID NO: 56) Catgaagcttgcatgcaaaacggtgacgttgaccgtcgacggaaccgcgatgcgggtgaccacgatgaaatcgcgggtgatcgacatcgtcgaagagaacgggttctcagtcgacgaccgcgacgacctgtatcccgcggccggcgtgcaggtccatgacgccgacaccatcgtgctgcggcgtagccgtccgctgcagatctcgctggatggtcacgacgctaagcaggtgtggacgaccgcgtcgacggtggacgaggcgctggcccaactcgcgatgaccgacacggcgccggccgcggcttctcgcgccagccgcgtcccgctgtccgggatggcgctaccggtcgtcagcgccaagacggtgcagctcaacgacggcgggttggtgcgcacggtgcacttgccggcccccaatgtcgcggggctgctgagtgcggccggcgtgccgctgttgcaaagcgaccacgtggtgcccgccgcgacggccccgatcgtcgaaggcatgcagatccaggtgacccgcaatcggatcaagaaggtcaccgagcggctgccgctgccgccgaacgcgcgtcgtgtcgaggacccggagatgaacatgagccgggaggtcgtcgaagacccgggggttccggggacccaggatgtgacgttcgcggtagctgaggtcaacggcgtcgagaccggccgtttgcccgtcgccaacgtcgtggtgaccccggcccacgaagccgtggtgcgggtgggcaccaagcccggtaccgaggtgcccccggtgatcgacggaagcatctgggacgcgatcgccggctgtgaggccggtggcaactgggcgatcaacaccggcaacgggtattacggtggtgtgcagtttgaccagggcacctgggaggccaacggcgggctgcggtatgcaccccgcgctgacctcgccacccgcgaagagcagatcgccgttgccgaggtgacccgactgcgtcaaggttggggcgcctggccggtatgtgctgcacgagcgggtgcgcgc ggatcc atgacagagcagcagtggaatttcgcgggtatcgaggccgcggcaagcgcaatccagggaaatgtcacgtccattcattccctccttgacgaggggaagcagtccctgaccaagctcgcagcggcctggggcggtagcggttcggaggcgtaccagggtgtccagcaaaaatgggacgccacggctaccgagctgaacaacgcgctgcagaacctggcgcggacgatcagcgaagccggtcaggcaatggcttcgaccgaaggcaacgtcactgggatgttcgcagaattc atgattgcgactacccgtgatcgtgagggcgcgaccatgatcacgttccgtctgcgtctgccgtgtcgcaccattttgcgcgtgttttcgcgtaacccgctggtccgcggtaccgaccgtctggaggccgttgtcatgctgctggcggttaccgtgagcctgctgacgatcccattcgcagcggcagctggcacggccgtccaagacagccgtagccatgtgtatgctcaccaggctcaaacccgtcacccggctactgccactgttatcgatcacgaaggcgtgattgactccaataccacggcaacctccgcaccgcctcgcaccaagattacggttcctgcgcgttgggtggtgaatggtattgaacgcagcggcgaagttaatgccaaaccgggtaccaaaagcggtgaccgtgtgggcatctgggtcgatagcgccggtcagctggtcgacgagccggcaccgccagcgcgtgcgatcgccgatgcggcgctggctgccctgggtctgtggctgagcgtggcagcggtcgccggtgcgttgctggcgctgacgcgcgcaattctgatccgcgttcgcaatgcgagctggcagcacgatattgatagcctgttttgcacccaacgt gagctc atgaccacggcgcgtgatatcatgaatgcgggtgtcacctgtgttggcgagcacgaaacgttgaccgcagcagcacagtacatgcgcgaacatgatatcggcgcattgccgatttgcggcgacgatgatcgtctgcacggtatgctgaccgaccgcgatatcgttatcaagggtctggccgcaggcttggacccgaacaccgcgaccgccggtgaactggcacgtgacagcatctattacgtcgacgcgaacgccagcattcaagagatgctgaacgtgatggaagagcatcaggtgcgtcgtgtcccggttatcagcgaacatcgtctggttggtatcgttaccgaagccgacatcgcacgtcacctgccggagcacgcgattgttcagttcgtgaaagcgatttgcagcccgatggcgttggcgtctcgtcaaaagggcgacacaaaatttattctaaatgcatga  (SEQ ID NO: 57)MKLACKTVTLTVDGTAMRVTTMKSRVIDIVEENGFSVDDRDDLYPAAGVQVHDADTIVLRRSRPLQISLDGHDAKQVWTTASTVDEALAQLAMTDTAPAAASRASRVPLSGMALPVVSAKTVQLNDGGLVRTVHLPAPNVAGLLSAAGVPLLQSDHVVPAATAPIVEGMQIQVTRNRIKKVTERLPLPPNARRVEDPEMNMSREVVEDPGVPGTQDVTFAVAEVNGVETGRLPVANVVVTPAHEAVVRVGTKPGTEVPPVIDGSTWDAIAGCEAGGNWAINTGNGYYGGVQFDQGTWEANGGLRYAPRADLATREEQIAVAEVTRLRQGWGAWPVCAARAGARGSMTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWDATATELNNALQNLARTISEAGQAMASTEGNVTGMFAEFMIATTRDREGATMFTFRLRLPCRTILRVFSRNPLVRGTDRLEAVVMLLAVTVSLLTIPFAAAAGTAVQDSRSHVYAHQAQTRHPATATVIDHEGVIDSNTTATSAPPRTKITVPARWVVNGIERSGEVNAKPGTKSGDRVGIWVDSAGQLVDEPAPPARAIADAALAALGLWLSVAAVAGALLALTRAILIRVRNASWQHDIDSLFCTQRELMTTARDTMNAGVTCVGEHETLTAAAQYMREHDIGALPICGDDDRLHGMLTDRDIVIKGLAAGLDPNTATAGELARDSIYYVDANASIQEMLNVMEEHQVRRVPVISEHRLVGIVTEADIARHLPEHAIVQFVKAICSPMALASRQKGDTKFILNA (SEQ ID NO: 58) Datgtttagccgtcctggcctgccagttgaatacctgcaagttccgagcccgtccatgggtcgtgacattaaggtgcagttccagagcggcggtaacaatagcccggctgtgtacctgctggacggtctgcgtgcgcaggatgattacaacggctgggacatcaataccccggcatttgagtggtattaccagtcgggtctgagcattgtgatgccggttggcggtcaaagcagcttctatagcgattggtacagcccggcatgcggcaaggctggttgccaaacctacaagtgggaaactttcttgaccagcgagctgccgcaatggttgagcgccaaccgtgcggtcaaaccgaccggtagcgctgctattggcctgtccatggccggcagcagcgcgatgatcttggcggcataccatccgcagcagtttatctacgccggtagcctgagcgcattgctggacccgagccaaggcatgggtccgagcctgattggtctggcaatgggtgacgcaggtggttacaaagcggccgatatgtggggcccatctagcgacccggcatgggagcgtaatgacccgacccagcaaattccgaaactggtggcgaataacacgcgcctgtgggtctactgtggcaatggtacgccgaacgagctgggtggcgcgaatatccctgcggagtttctggaaaactttgttcgcagcagcaacctgaaattccaggacgcgtataacgcagccggtggtcacaatgcggttttcaatttcccgccaaatggcactcatagctgggagtactggggtgcgcagttgaacgcaatgaaaggcgatctgcaatcctctctgggtgcgggcggatcc atgacagagcagcagtggaatttcgcgggtatcgaggccgcggcaagcgcaatccagggaaatgtcacgtccattcattccctccttgacgaggggaagcagtccctgaccaagctcgcagcggcctggggcggtagcggttcggaggcgtaccagggtgtccagcaaaaatgggacgccacggctaccgagctgaacaacgcgctgcagaacctggcgcggacgatcagcgaagccggtcaggcaatggcttcgaccgaaggcaacgtcactgggatgttcgca gaattc atgattgcgactacccgtgatcgtgagggcgcgaccatgatcacgttccgtctgcgtctgccgtgtcgcaccattttgcgcgtgttttcgcgtaacccgctggtccgcggtaccgaccgtctggaggcccccggggtccaagacagccgtagccatgtgtatgctcaccaggctcaaacccgtcacccggctactgccactgttatcgatcacgaaggcgtgattgactccaataccacggcaacctccgcaccgcctcgcaccaagattacggttcctgcgcgttgggtggtgaatggtattgaacgcagcggcgaagttaatgccaaaccgggtaccaaaagcggtgaccgtgtgggcatctgggtcgatagcgccggtcagctggtcgacgagccggcaccgccagcgcgtgcgatcgccgattctagacgcgcaattctgatccgcgttcgcaatgcgagctggcagcacgatattgatagcctgttttgcacccaacgt gagctc atgaccacggcgcgtgatatcatgaatgcgggtgtcacctgtgttggcgagcacgaaacgttgaccgcagcagcacagtacatgcgcgaacatgatatcggcgcattgccgatttgcggcgacgatgatcgtctgcacggtatgctgaccgaccgcgatatcgttatcaagggtctggccgcaggcttggacccgaacaccgcgaccgccggtgaactggcacgtgacagcatctattacgtcgacgcgaacgccagcattcaagagatgctgaacgtgatggaagagcatcaggtgcgtcgtgtcccggttatcagcgaacatcgtctggttggtatcgttaccgaagccgacatcgcacgtcacctgccggagcacgcgattgttcagttcgtgaaagcgatttgcagcccgatggcgttggcgtct aagctt ttgctgggcctgagcaccattagcagcaaagcggatgacatcgactgggatgcgattgcgcagtgtgagagcggtggcaattgggcagcgaataccggcaatggcctgtacggcggtctgcagatctcccaggcgacgtgggacagcaatggtggcgtcggcagcccggctgccgcgtccccacaacaacagatcgaggtggcagataacattatgaaaacgcagggtccgggtgcttggccaaaatgctccagctgcagccagggtgacgcaccgctgggcagcctgacccacattctgacgttcctggcagcggaaaccggtggttgtagcggtagccgcgatgac (SEQ ID NO: 59)atgttctccaggcccggcctgcctgtcgagtatctgcaggtcccctccccctccatgggcagagacatcaaggtgcagttccaatccggaggcaacaacagccccgccgtgtatctcctcgacggcctgagggctcaggacgactacaacggctgggacatcaacacccccgccttcgagtggtactaccagtccggactgagcatcgtcatgcccgtgggcggccagagctccttctacagcgactggtatagccctgcctgcggcaaagccggatgccagacctacaagtgggagacctttctgaccagcgaactgccccagtggctgtccgccaatagggccgtcaaacctaccggctccgctgccatcggactcagcatggccggaagctccgctatgatcctggccgcctaccacccccagcaatttatctacgctggcagcctgtccgctctgctggatcctagccaaggcatgggccctagcctcattggcctggccatgggcgatgctggcggctataaggccgccgatatgtggggccctagctccgatcctgcctgggagaggaatgaccccacccagcagatccccaagctggtggccaacaacacaaggctctgggtgtactgcggcaatggcacccccaacgaactgggcggagccaacattcccgccgagttcctggagaacttcgtcaggagcagcaacctgaagttccaggacgcctacaatgccgccggaggccacaacgctgtgttcaacttccctcccaacggcacccacagctgggagtattggggcgctcagctgaacgccatgaaaggcgacctccagagctccctgggagctggacccggg accgagcagcagtggaacttcgccggcatcgaagctgccgctagcgccatccaaggcaacgtgaccagcatccacagcctgctggacgagggcaagcagagcctgaccaagctggctgctgcttggggcggatccggaagcgaagcctaccagggcgtgcagcagaagtgggacgccacagccaccgagctgaacaacgccctgcagaacctcgccagaaccatcagcgaggccggacaggctatggccagcacagagggcaatgtgaccggcatgttcgcc ttcgaa atcgccaccaccagggacagggaaggcgctaccatgatcaccttcaggctgaggctcccctgcaggaccatcctgagggtgttcagcaggaaccccctggtgaggggcaccgacagactggaagccgtgcaggacagcaggagccacgtgtatgcccaccaggctcagaccaggcaccctgctaccgccaccgtgatcgaccacgagggcgtgatcgactccaacaccaccgccaccagcgctcctcccagaaccaagatcacagtgcccgccaggtgggtggtgaacggcatcgagaggagcggcgaggtgaacgccaagcctggaaccaagagcggcgacagggtgggcatttgggtcgatagcgccggccagctggtggatgaacctgctccccctgccagagccatcgccgatagggccatcctgatcagggtgaggaacgccagctggcagcacgacatcgacagcctgttctgcacccaaagg cgatcg acaacagccagggacatcatgaacgccggcgtgacctgcgtgggagagcatgaaaccctcaccgccgccgcccaatacatgagggagcacgacatcggcgccctgcccatctgtggagacgacgacaggctgcacggcatgctgaccgacagggacatcgtgatcaagggcctggctgccggcctcgatcctaacaccgctacagccggcgagctggccagagacagcatctactacgtggacgccaacgccagcatccaggagatgctcaacgtgatggaggagcaccaggtgagaagggtgcctgtgatcagcgagcacaggctggtgggcatcgtgaccgaggccgatatcgctaggcacctgcccgagcacgccatcgtgcagttcgtgaaggccatctgcagccccatggctctggccagc ggcg cgcc cacccccggactcctcaccacagctggagctggcaggcccagagacagatgcgccaggatcgtgtgcaccgtgttcatcgagaccgccgtggtggctaccatgttcgtggccctgctgggcctgagcaccatcagcagcaaggccgacgacatcgactgggacgccatcgcccagtgtgaatccggcggaaactgggccgccaataccggcaatggcctgtacggcggcctgcagatcagccaggctacctgggactccaacggaggagtgggaagccctgccgctgcttcccctcagcagcagatcgaggtggccgacaacatcatgaagacccaaggccctggcgcctggcctaagtgttccagctgtagccagggcgatgctcctctgggcagcctgacccacatcctgacctttctcgccgccgagacaggcggatgtagcggaagcagggacgactaatgatag (SEQ ID NO: 60)MFSRPGLPVEYLQVPSPSMGRDIKVQFQSGGNNSPAVYLLDGLRAQDDYNGWDINTPAFEWYYQSGLSIVMPVGGQSSFYSDWYSPACGKAGCQTYKWETFLTSELPQWLSANRAVKPTGSAAIGLSMAGSSAMILAAYHPQQFIYAGSLSALLDPSQGMGPSLIGLAMGDAGGYKAADMWGPSSDPAWERNDPTQQIPKLVANNTRLWVYCGNGTPNELGGANIPAEFLENFVRSSNLKFQDAYNAAGGHNAVFNFPPNGTHSWEYWGAQLNAMKGDLQSSLGAGGSMTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWDATATELNNALQNLARTISEAGQAMASTEGNVTGMFAEFMIATTRDREGATMITFRLRLPCRTILRVFSRNPLVRGTDRLEAPGVQDSRSHVYAHQAQTRHPATATVIDHEGVTDSNTTATSAPPRTKITVPARWVVNGIERSGEVNAKPGTKSGDRVGIWVDSAGQLVDEPAPPARAIADSRRAILIRVRNASWQHDIDSLFCTQRELMTTARDIMNAGVTCVGEHETLTAAAQYMREHDIGALPICGDDDRLHGMLTDRDIVIKGLAAGLDPNTATAGELARDSIYYVDANASIQEMLNVMEEHQVRRVPVISEHRLVGIVTEADIARHLPEHAIVQFVKAICSPMALASKLLLGLSTISSKADDIDWDAIAQCESGGNWAANTGNGLYGGLQISQATWDSNGGVGSPAAASPQQQIEVADNIMKTQGPGAWPKCSSCSQGDAPLGSLTHILTFLAAETGGCSGSRDD (SEQ ID NO: 61)MFSRPGLPVEYLQVPSPSMGRDIKVQFQSGGNNSPAVYLLDGLRAQDDYNGWDINTPAFEWYYQSGLSIVMPVGGQSSFYSDWYSPACGKAGCQTYKWETFLTSELPQWLSANRAVKPTGSAAIGLSMAGSSAMILAAYHPQQFTYAGSLSALLDPSQGMGPSLIGLAMGDAGGYKAADMWGPSSDPAWERNDPTQQIPKLVANNTRLWVYCGNGTPNELGGANIPAEFLENFVRSSNLKFQDAYNAAGGHNAVFNFPPNGTHSWEYWGAQLNAMKGDLQSSLGAGPGTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWDATATELNNALQNLARTTSEAGQAMASTEGNVTGMFAFEIATTRDREGATMITFRLRLPCRTILRVFSRNPLVRGTDRLEAVQDSRSHVYAHQAQTRHPATATVIDHEGVIDSNTTATSAPPRTKITVPARWVVNGIERSGEVNAKPGTKSGDRVGIWVDSAGQLVDEPAPPARAIADRAILIRVRNASWQHDIDSLFCTQRRSTTARDIMNAGVTCVGEHETLTAAAQYMREHDIGALPICGDDDRLHGMLTDRDIVIKGLAAGLDPNTATAGELARDSIYYVDANASIQEMLNVMEEHQVRRVPVISEHRLVGIVTEADIARHLPEHAIVQFVKAICSPMALASGAPTPGLLTTAGAGRPRDRCARJVCTVFIETAVVATMFVALLGLSTISSKADDIDWDAIAQCESGGNWAANTGNGLYGGLQISQATWDSNGGVGSPAAASPQQQTEVADNIMKTQGPGAWPKCSSCSQGDAPLGSLTHTLTFLAAETGG CSGSRDD (SEQ ID NO: 62)E atggattttgcgctgctgccgccggaagtgaacagcgcgcgcatgtataccggcccgggcgcgggcagcctgctggcggcggcgggcggctgggatagcctggcggcggaactggcgaccaccgcggaagcgtatggcagcgtgctgagcggcctggcggcgctgcattggcgcggcccggcggcggaaagcatggcggtgagcgccgtatattggctggctgtataccaccgcggaaaaaacccagcagaccgcgattcaggcgcgcgcggcggcgctggcgtttgaacaggcgtatgcgatgaccctgccgccgccggtggtggcggcgaaccgcattcagctgctggcgctgattgcgaccaacttttttggccagaacaccgcggcgattgcggcgaccgaagcgcagtatgcggaaatgtgggcgcaggatgcggcggcgatgtatggctatgcgaccgcgagcgcggcggcggcgctgctgaccccgtttagcccgccgcgccagaccaccaacccggcgggcctgaccgcgcaggcggcggcggtgagccaggcgaccgatccgctgagcctgctgattgaaaccgtgacccaggcgctgcaggcgctgaccattccgagctttattccggaagattttacctttctggatgcgatttttgcgggctatgcgaccgtgggcgtgacccaggatgtggaaagctttgtggcgggcaccattggcgcggaaagcaacctgggcctgctgaacgtgggcgatgaaaacccggcggaagtgaccccgggcgattttggcattggcgaactggtgagcgcgaccagcccgggcggcggcgtgagcgcgagcggcgcgggcggcgcggcgagcgtgggcaacaccgtgctggcgagcgtgggccgcgcgaacagcattggccagctgagcgtgccgccgagctgggcggcgccgagcacccgcccggtgagcgcgctgagcccggcgggcctgaccaccctgccgggcaccgatgtggcggaacatggcatgccgggcgtgccgggcgtgccggtggcggcgggccgcgcgagcggcgtgctgccgcgctatggcgtgcgcctgaccgtgatggcgcatccgccggcggcgggc gaattc atgattgcgactacccgtgatcgtgagggcgcgaccatgatcacgttccgtctgcgtctgccgtgtcgcaccattttgcgcgtgttttcgcgtaacccgctggtccgcggtaccgaccgtctggaggccgttgtcatgctgctggcggttaccgtgagcctgctgacgatcccattcgcagcggcagctggcacggccgtccaagacagccgtagccatgtgtatgctcaccaggctcaaacccgtcacccggctactgccactgttatcgatcacgaaggcgtgattgactccaataccacggcaacctccgcaccgcctcgcaccaagattacggttcctgcgcgttgggtggtgaatggtattgaacgcagcggcgaagttaatgccaaaccgggtaccaaaagcggtgaccgtgtgggcatctgggtcgatagcgccggtcagctggtcgacgagccggcaccgccagcgcgtgcgatcgccgatgcggcgctggctgccctgggtctgtggctgagcgtggcagcggtcgccggtgcgttgctggcgctgacgcgcgcaattctgatccgcgttcgcaatgcgagctggcagcacgatattgatagcctgttttgcacccaacgt gagctc atgtccacgcaacgaccgaggcactccggtattcgggctgttggcccctacgcatgggccggccgatgtggtcggataggcaggtggggggtgcaccaggaggcgatgatgaatctagcgatatggcacccgcgcaaggtgcaatccgccaccatctatcaggtgaccgatcgctcgcacgacgggcgcacagcacgggtgcctggtgacgagatcactagcaccgtgtccggttggttgtcggagttgggcacccaaagcccgttggccgatgagcttgcgcgtgcggtgcggatcggcgactggcccgctgcgtacgcaatcggtgagcacctgtccgttgagattgccgttgcggtc aagctt ttgctgggcctgagcaccattagcagcaaagcggatgacatcgactgggatgcgattgcgcagtgtgagagcggtggcaattgggcagcgaataccggcaatggcctgtacggcggtctgcagatctcccaggcgacgtgggacagcaatggtggcgtcggcagcccggctgccgcgtccccacaacaacagatcgaggtggcagataacattatgaaaacgcagggtccgggtgcttggccaaaatgctccagctgcagccagggtgacgcaccgctgggcagcctgacccacattctgacgttcctggcagcggaaaccggtggttgtagcggtagccgcgatgactga  (SEQ ID NO: 63)MDFALLPPEVNSARMYTGPGAGSLLAAAGGWDSLAAELATTAEAYGSVLSGLAALHWRGPAAESMAVTAAPYIGWLYTTAEKTQQTAIQARAAALAFEQAYAMTLPPPVVAANRIQLLALIATNFFGQNTAAIAATEAQYAEMWAQDAAAMYGYATASAAAALLTPFSPPRQTTNPAGLTAQAAAVSQATDPLSLLIETVTQALQALTIPSFIPEDFTFLDAIFAGYATVGVTQDVESFVAGTIGAESNLGLLNVGDENPAEVTPGDFGIGELVSATSPGGGVSASGAGGAASVGNTVLASVGRANSIGQLSVPPSWAAPSTRPVSALSPAGLTTLPGTDVAEHGMPGVPGVPVAAGRASGVLPRYGVRLTVMAHPPAAGEFEFMIATTRDREGATMTTFRLRLPCRTILRVFSRNPLVRGTDRLEAVVMLLAVTVSLLTIPFAAAAGTAVQDSRSHVYAHQAQTRHPATATVIDHEGVIDSNTTATSAPPRTKITVPARWVVNGIERSGEVNAKPGTKSGDRVGIWVDSAGQLVDEPAPPARAIADAALAALGLWLSVAAVAGALLALTRAILIRVRNASWQHDIDSLFCTQRELMSTQRPRHSGIRAVGPYAWAGRCGRIGRWGVHQEAMMNLAIWHPRKVQSATIYQVTDRSHDGRTARVPGDEITSTVSGWLSELGTQSPLADELARAVRIGDWPAAYAIGEHLSVEIAVAVKLLLGLSTISSKADDIDWDAIAQCESGGNWAANTGNGLYGGLQISQATWDSNGGVGSPAAASPQQQIEVADNIMKTQGPGAWPKCSSCSQGDAPLGSLTHILTFLAAETGGCSGSRDD (SEQ ID NO: 64) Fatggattttgcgctgctgccgccggaagtgaacagcgcgcgcatgtataccggcccgggcgcgggcagcctgctggcggcggcgggcggctgggatagcctggcggcggaactggcgaccaccgcggaagcgtatggcagcgtgctgagcggcctggcggcgctgcattggcgcggcccggcggcggaaagcatggcggtgaccgcggcgccgtatattggctggctgtataccaccgcggaaaaaacccagcagaccgcgattcaggcgcgcgcggcggcgctggcgtttgaacaggcgtatgcgatgaccctgccgccgccggtggtggcggcgaaccgcattcagctgctggcgctgattgcgaccaacttttttggccagaacaccgcggcgattgcggcgaccgaagcgcagtatgcggaaatgtgggcgcaggatgcggcggcgatgtatggctatgcgaccgcgagcgcggcggcggcgctgctgaccccgtttagcccgccgcgccagaccaccaacccggcgggcctgaccgcgcaggcggcggcggtgagccaggcgaccgatccgctgagcctgctgattgaaaccgtgacccaggcgctgcaggcgctgaccattccgagctttattccggaagattttacctttctggatgcgatttttgcgggctatgcgaccgtgggcgtgacccaggatgtggaaagctttgtggcgggcaccattggcgcggaaagcaacctgggcctgctgaacgtgggcgatgaaaacccggcggaagtgaccccgggcgattttggcattggcgaactggtgagcgcgaccagcccgggcggcggcgtgagcgcgagcggcgcgggcggcgcggcgagcgtgggcaacaccgtgctggcgagcgtgggccgcgcgaacagcattggccagctgagcgtgccgccgagctgggcggcgccgagcacccgcccggtgagcgcgctgagcccggcgggcctgaccaccctgccgggcaccgatgtggcggaacatggcatgccgggcgtgccgggcgtgccggtggcggcgggccgcgcgagcggcgtgctgccgcgctatggcgtgcgcctgaccgtgatggcgcatccgccggcggcgggc gaattc atgattgcgactacccgtgatcgtgagggcgcgaccatgatcacgttccgtctgcgtctgccgtgtcgcaccattttgcgcgtgttttcgcgtaacccgctggtccgcggtaccgaccgtctggaggccgttgtcatgctgctggcggttaccgtgagcctgctgacgatcccattcgcagcggcagctggcacggccgtccaagacagccgtagccatgtgtatgctcaccaggctcaaacccgtcacccggctactgccactgttatcgatcacgaaggcgtgattgactccaataccacggcaacctccgcaccgcctcgcaccaagattacggttcctgcgcgttgggtggtgaatggtattgaacgcagcggcgaagttaatgccaaaccgggtaccaaaagcggtgaccgtgtgggcatctgggtcgatagcgccggtcagctggtcgacgagccggcaccgccagcgcgtgcgatcgccgatgcggcgctggctgccctgggtctgtggctgagcgtggcagcggtcgccggtgcgttgctggcgctgacgcgcgcaattctgatccgcgttcgcaatgcgagctggcagcacgatattgatagcctgttttgcacccaacgt gagctcatgtccacgcaacgaccgaggcactccggtattcgggctgttggcccctacgcatgggccggccgatgtggtcggataggcaggtggggggtgcaccaggaggcgatgatgaatctagcgatatggcacccgcgcaaggtgcaatccgccaccatctatcaggtgaccgatcgctcgcacgacgggcgcacagcacgggtgcctggtgacgagatcactagcaccgtgtccggttggttgtcggagttgggcacccaaagcccgttggccgatgagcttgcgcgtgcggtgcggatcggcgactggcccgctgcgtacgcaatcggtgagcacctgtccgttgagattgccgttgcggtc aagctt gcatgcaaaacggtgacgttgaccgtcgacggaaccgcgatgcgggtgaccacgatgaaatcgcgggtgatcgacatcgtcgaagagaacgggttctcagtcgacgaccgcgacgacctgtatcccgcggccggcgtgcaggtccatgacgccgacaccatcgtgctgcggcgtagccgtccgctgcagatctcgctggatggtcacgacgctaagcaggtgtggacgaccgcgtcgacggtggacgaggcgctggcccaactcgcgatgaccgacacggcgccggccgcggcttctcgcgccagccgcgtcccgctgtccgggatggcgctaccggtcgtcagcgccaagacggtgcagctcaacgacggcgggttggtgcgcacggtgcacttgccggcccccaatgtcgcggggctgctgagtgcggccggcgtgccgctgttgcaaagcgaccacgtggtgcccgccgcgacggccccgatcgtcgaaggcatgcagatccaggtgacccgcaatcggatcaagaaggtcaccgagcggctgccgctgccgccgaacgcgcgtcgtgtcgaggacccggagatgaacatgagccgggaggtcgtcgaagacccgggggttccggggacccaggatgtgacgttcgcggtagctgaggtcaacggcgtcgagaccggccgtttgcccgtcgccaacgtcgtggtgaccccggcccacgaagccgtggtgcgggtgggcaccaagcccggtaccgaggtgcccccggtgatcgacggaagcatctgggacgcgatcgccggctgtgaggccggtggcaactgggcgatcaacaccggcaacgggtattacggtggtgtgcagtttgaccagggcacctgggaggccaacggcgggctgcggtatgcaccccgcgctgacctcgccacccgcgaagagcagatcgccgttgccgaggtgacccgactgcgtcaaggttggggcgcctggccggtatgtgctgcacgagcgggtgcgcgctga (SEQ ID NO: 65) MDFALLPPEVNSARMYTGPGAGSLLAAAGGWDSLAAELATTAEAYGSVLSGLAALHWRGPAAESMAVTAAPYIGWLYTTAEKTQQTAIQARAAALAFEQAYAMTLPPPVVAANRIQLLALIATNFFGQNTAAIAATEAQYAEMWAQDAAAMYGYATASAAAALLTPFSPPRQTTNPAGLTAQAAAVSQATDPLSLLIETVTQALQALTIPSFIPEDFTFLDAIFAGYATVGVTQDVESFVAGTIGAESNLGLLNVGDENPAEVTPGDFGIGELVSATSPGGGVSASGAGGAASVGNTVLASVGRANSIGQLSVPPSWAAPSTRPVSALSPAGLTTLPGTDVAEHGMPGVPGVPVAAGRASGVLPRYGVRLTVMAHPPAAGEFMIATTRDREGATMITFRLRLPCRTILRVFSRNPLVRGTDRLEAVVMLLAVTVSLLTIPFAAAAGTAVQDSRSHVYAHQAQTRHPATATVIDHEGVIDSNTTATSAPPRTKITVPARWVVNGIERSGEVNAKPGTKSGDRVGIWVDSAGQLVDEPAPPARAIADAALAALGLWLSVAAVAGALLALTRAILIRVRNASWQHDIDSLFCTQRELMSTQRPRHSGIRAVGPYAWAGRCGRIGRWGVHQEAMMNLAIWHPRKVQSATIYQVTDRSHDGRTARVPGDEITSTVSGWLSELGTQSPLADELARAVRIGDWPAAYAIGEHLSVEIAVAVKLACKTVTLTVDGTAMRVTTMKSRVIDIVEENGFSVDDRDDLYPAAGVQVHDADTIVLRRSRPLQISLDGHDAKQVWTTASTVDEALAQLAMTDTAPAAASRASRVPLSGMALPVVSAKTVQLNDGGLVRTVHLPAPNVAGLLSAAGVPLLQSDHVVPAATAPIVEGMQIQVTRNRIKKVTERLPLPPNARRVEDPEMNMSREVVEDPGVPGTQDVTFAVAEVNGVETGRLPVANVVVTPAHEAVVRVGTKPGTEVPPVIDGSIWDAIAGCEAGGNWAINTGNGYYGGVQFDQGTWEANGGLRYAPRADLATREEQIAVAEVTRLRQGWGAWPVCAARAGAR (SEQ ID NO: 66) Gatgcgtgcgactgtgggtctggttgaggcgattggcattcgcgagctgcgccaacatgccagccgttacttggctcgtgtcgaggcgggtgaagaactgggcgtgacgaataagggtcgtctggtcgcccgtctgattccggttcaggcagctgagcgttctcgcgaggcgctgattgaatccggcgtcctgatcccggctcgccgtccgcaaaacctgctggacgtgacggcggagccagctcgtggtcgcaaacgcacgctgtctgatgtcctgaacgaaatgcgcgacgagcag gaattc atgattgcgactacccgtgatcgtgagggcgcgaccatgatcacgttccgtctgcgtctgccgtgtcgcaccattttgcgcgtgttttcgcgtaacccgctggtccgcggtaccgaccgtctggaggccgttgtcatgctgctggcggttaccgtgagcctgctgacgatcccattcgcagcggcagctggcacggccgtccaagacagccgtagccatgtgtatgctcaccaggctcaaacccgtcacccggctactgccactgttatcgatcacgaaggcgtgattgactccaataccacggcaacctccgcaccgcctcgcaccaagattacggttcctgcgcgttgggtggtgaatggtattgaacgcagcggcgaagttaatgccaaaccgggtaccaaaagcggtgaccgtgtgggcatctgggtcgatagcgccggtcagctggtcgacgagccggcaccgccagcgcgtgcgatcgccgatgcggcgctggctgccctgggtctgtggctgagcgtggcagcggtcgccggtgcgttgctggcgctgacgcgcgcaattctgatccgcgttcgcaatgcgagctggcagcacgatattgatagcctgttttgcacccaacgt g a g ctc atgaccacggcgcgtgatatcatgaatgcgggtgtcacctgtgttggcgagcacgaaacgttgaccgcagcagcacagtacatgcgcgaacatgatatcggcgcattgccgatttgcggcgacgatgatcgtctgcacggtatgctgaccgaccgcgatatcgttatcaagggtctggccgcaggcttggacccgaacaccgcgaccgccggtgaactggcacgtgacagcatctattacgtcgacgcgaacgccagcattcaagagatgctgaacgtgatggaagagcatcaggtgcgtcgtgtcccggttatcagcgaacatcgtctggttggtatcgttaccgaagccgacatcgcacgtcacctgccggagcacgcgattgttcagttcgtgaaagcgatttgcagcccgatggcgttggcgtctcgtcaaaagggcgacacaaaatttattctaaatgca aagctt gcatgcaaaacggtgacgttgaccgtcgacggaaccgcgatgcgggtgaccacgatgaaatcgcgggtgatcgacatcgtcgaagagaacgggttctcagtcgacgaccgcgacgacctgtatcccgcggccggcgtgcaggtccatgacgccgacaccatcgtgctgcggcgtagccgtccgctgcagatctcgctggatggtcacgacgctaagcaggtgtggacgaccgcgtcgacggtggacgaggcgctggcccaactcgcgatgaccgacacggcgccggccgcggcttctcgcgccagccgcgtcccgctgtccgggatggcgctaccggtcgtcagcgccaagacggtgcagctcaacgacggcgggttggtgcgcacggtgcacttgccggcccccaatgtcgcggggctgctgagtgcggccggcgtgccgctgttgcaaagcgaccacgtggtgcccgccgcgacggccccgatcgtcgaaggcatgcagatccaggtgacccgcaatcggatcaagaaggtcaccgagcggctgccgctgccgccgaacgcgcgtcgtgtcgaggacccggagatgaacatgagccgggaggtcgtcgaagacccgggggttccggggacccaggatgtgacgttcgcggtagctgaggtcaacggcgtcgagaccggccgtttgcccgtcgccaacgtcgtggtgaccccggcccacgaagccgtggtgcgggtgggcaccaagcccggtaccgaggtgcccccggtgatcgacggaagcatctgggacgcgatcgccggctgtgaggccggtggcaactgggcgatcaacaccggcaacgggtattacggtggtgtgcagtttgaccagggcacctgggaggccaacggcgggctgcggtatgcaccccgcgctgacctcgccacccgcgaagagcagatcgccgttgccgaggtgacccgactgcgtcaaggttggggcgcctggccggtatgtgctgcacgagcgggtgcgcgctga (SEQ ID NO: 67) MRATVGLVEAIGIRELRQHASRYLARVEAGEELGVTNKGRLVARLIPVQAAERSREALIESGVLIPARRPQNLLDVTAEPARGRKRTLSDVLNEMRDEQEFMIATTRDREGATMITFRLRLPCRTILRVFSRNPLVRGTDRLEAVVMLLAVTVSLLTIPFAAAAGTAVQDSRSHVYAHQAQTRHPATATVIDHEGVIDSNTTATSAPPRTKITVPARWVVNGTERSGEVNAKPGTKSGDRVGIWVDSAGQLVDEPAPPARAIADAALAALGLWLSVAAVAGALLALTRAILIRVRNASWQHDIDSLFCTQRELMTTARDIMNAGVTCVGEHETLTAAAQYMREHDIGALPICGDDDRLHGMLTDRDIVIKGLAAGLDPNTATAGELARDSIYYVDANASIQEMLNVMEEHQVRRVPVISEHRLVGIVTEADIARHLPEHAIVQFVKAICSPMALASRQKGDTKFILNAKLACKTVTLTVDGTAMRVTTMKSRVIDIVEENGFSVDDRDDLYPAAGVQVHDADTIVLRRSRPLQISLDGHDAKQVWTTASTVDEALAQLAMTDTAPAAASRASRVPLSGMALPWSAKTVQLNDGGLVRTVHLPAPNVAGLLSAAGVPLLQSDHVVPAATAPIVEGMQIQVTRNRIKKVTERLPLPPNARRVEDPEMNMSREVVEDPGVPGTQDVTFAVAEVNGVETGRLPVANVVVTPAHEAVVRVGTKPGTEVPPVIDGSIWDAIAGCEAGGNWAINTGNGYYGGVQFDQGTWEANGGLRYAPRADLATREEQIAVAEVTRLRQGWGAWPVCAARAGAR (SEQ ID NO: 68) Hatgcgtgcgactgtgggtctggttgaggcgattggcattcgcgagctgcgccaacatgccagccgttacttggctcgtgtcgaggcgggtgaagaactgggcgtgacgaataagggtcgtctggtcgcccgtctgattccggttcaggcagctgagcgttctcgcgaggcgctgattgaatccggcgtcctgatcccggctcgccgtccgcaaaacctgctggacgtgacggcggagccagctcgtggtcgcaaacgcacgctgtctgatgtcctgaacgaaatgcgcgacgagcag gaattc atgattgcgactacccgtgatcgtgagggcgcgaccatgatcacgttccgtctgcgtctgccgtgtcgcaccattttgcgcgtgttttcgcgtaacccgctggtccgcggtaccgaccgtctggaggccgttgtcatgctgctggcggttaccgtgagcctgctgacgatcccattcgcagcggcagctggcacggccgtccaagacagccgtagccatgtgtatgctcaccaggctcaaacccgtcacccggctactgccactgttatcgatcacgaaggcgtgattgactccaataccacggcaacctccgcaccgcctcgcaccaagattacggttcctgcgcgttgggtggtgaatggtattgaacgcagcggcgaagttaatgccaaaccgggtaccaaaagcggtgaccgtgtgggcatctgggtcgatagcgccggtcagctggtcgacgagccggcaccgccagcgcgtgcgatcgccgatgcggcgctggctgccctgggtctgtggctgagcgtggcagcggtcgccggtgcgttgctggcgctgacgcgcgcaattctgatccgcgttcgcaatgcgagctggcagcacgatattgatagcctgttttgcacccaacgt gagctc atgaccacggcgcgtgatatcatgaatgcgggtgtcacctgtgttggcgagcacgaaacgttgaccgcagcagcacagtacatgcgcgaacatgatatcggcgcattgccgatttgcggcgacgatgatcgtctgcacggtatgctgaccgaccgcgatatcgttatcaagggtctggccgcaggcttggacccgaacaccgcgaccgccggtgaactggcacgtgacagcatctattacgtcgacgcgaacgccagcattcaagagatgctgaacgtgatggaagagcatcaggtgcgtcgtgtcccggttatcagcgaacatcgtctggttggtatcgttaccgaagccgacatcgcacgtcacctgccggagcacgcgattgttcagttcgtgaaagcgatttgcagcccgatggcgttggcgtctcgtcaaaagggcgacacaaaatttattctaaatgca aagctt ttgctgggcctgagcaccattagcagcaaagcggatgacatcgactgggatgcgattgcgcagtgtgagagcggtggcaattgggcagcgaataccggcaatggcctgtacggcggtctgcagatctcccaggcgacgtgggacagcaatggtggcgtcggcagcccggctgccgcgtccccacaacaacagatcgaggtggcagataacattatgaaaacgcagggtccgggtgcttggccaaaatgctccagctgcagccagggtgacgcaccgctgggcagcctgacccacattctgacgttcctggcagcggaaaccggttgtagcggtagccgcgatgactga (SEQ ID NO: 69)MRATVGLVEAIGIRELRQHASRYLARVEAGEELGVTNKGRLVARLIPVQAAERSREALIESGVLIPARRPQNLLDVTAEPARGRKRTLSDVLNEMRDEQEFMIATTRDREGATMITFRLRLPCRTILRVFSRNPLVRGTDRLEAVVMLLAVTVSLLTIPFAAAAGTAVQDSRSHVYAHQAQTRHPATATVIDHEGVIDSNTTATSAPPRTKITVPARWVVNGIERSGEVNAKPGTKSGDRVGIWVDSAGQLVDEPAPPARAIADAALAALGLWLSVAAVAGALLALTRAILIRVRNASWQHDIDSLFCTQRELMTTARDIMNAGVTCVGEHETLTAAAQYMREHDIGALPICGDDDRLHGMLTDRDIVIKGLAAGLDPNTATAGELARDSIYYVDANASIQEMLNVMEEHQVRRVPVISEHRLVGIVTEADIARHLPEHAIVQFVKAICSPMALASRQKGDTKFILNAKLLLGLSTISSKADDIDWDAIAQCESGGNWAANTGNGLYGGLQISQATWDSNGGVGSPAAASPQQQIEVADNIMKTQGPGAWPKCSSCSQGDAPLGSLTHILTFLAAETGGCSGSRDD (SEQ ID NO: 70) Iatggattttgcgctgctgccgccggaagtgaacagcgcgcgcatgtataccggcccgggcgcgggcagcctgctggcggcggcgggcggctgggatagcctggcggcggaactggcgaccaccgcggaagcgtatggcagcgtgctgagcggcctggcggcgctgcattggcgcggcccggcggcggaaagcatggcggtgaccgcggcgccgtatattggctggctgtataccaccgcggaaaaaacccagcagaccgcgattcaggcgcgcgcggcggcgctggcgtttgaacaggcgtatgcgatgaccctgccgccgccggtggtggcggcgaaccgcattcagctgctggcgctgattgcgaccaacttttttggccagaacaccgcggcgattgcggcgaccgaagcgcagtatgcggaaatgtgggcgcaggatgcggcggcgatgtatggctatgcgaccgcgagcgcggcggcggcgctgctgaccccgtttagcccgccgcgccagaccaccaacccggcgggcctgaccgcgcaggcggcggcggtgagccaggcgaccgatccgctgagcctgctgattgaaaccgtgacccaggcgctgcaggcgctgaccattccgagctttattccggaagattttacctttctggatgcgatttttgcgggctatgcgaccgtgggcgtgacccaggatgtggaaagctttgtggcgggcaccattggcgcggaaagcaacctgggcctgctgaacgtgggcgatgaaaacccggcggaagtgaccccgggcgattttggcattggcgaactggtgagcgcgaccagcccgggcggcggcgtgagcgcgagcggcgcgggcggcgcggcgagcgtgggcaacaccgtgctggcgagcgtgggccgcgcgaacagcattggccagctgagcgtgccgccgagctgggcggcgccgagcacccgcccggtgagcgcgctgagcccggcgggcctgaccaccctgccgggcaccgatgtggcggaacatggcatgccgggcgtgccgggcgtgccggtggcggcgggccgcgcgagcggcgtgctgccgcgctatggcgtgcgcctgaccgtgatggcgcatccgccggcggcgggcgaatttatgacagagcagcagtggaatttcgcgggtatcgaggccgcggcaagcgcaatccagggaaatgtcacgtccattcattccctccttgacgaggggaagcagtccctgaccaagctcgcagcggcctggggcggtagcggttcggaggcgtaccagggtgtccagcaaaaatgggacgccacggctaccgagctgaacaacgcgctgcagaacctggcgcggacgatcagcgaagccggtcaggcaatggcttcgaccgaaggcaacgtcactgggatgttcgca gaattc atgattgcgactacccgtgatcgtgagggcgcgaccatgatcacgttccgtctgcgtctgccgtgtcgcaccattttgcgcgtgttttcgcgtaacccgctggtccgcggtaccgaccgtctggaggccgttgtcatgctgctggcggttaccgtgagcctgctgacgatcccattcgcagcggcagctggcacggccgtccaagacagccgtagccatgtgtatgctcaccaggctcaaacccgtcacccggctactgccactgttatcgatcacgaaggcgtgattgactccaataccacggcaacctccgcaccgcctcgcaccaagattacggttcctgcgcgttgggtggtgaatggtattgaacgcagcggcgaagttaatgccaaaccgggtaccaaaagcggtgaccgtgtgggcatctgggtcgatagcgccggtcagctggtcgacgagccggcaccgccagcgcgtgcgatcgccgatgcggcgctggctgccctgggtctgtggctgagcgtggcagcggtcgccggtgcgttgctggcgctgacgcgcgcaattctgatccgcgttcgcaatgcgagctggcagcacgatattgatagcctgttttgcacccaacgt gagctc atgaccacggcgcgtgatatcatgaatgcgggtgtcacctgtgttggcgagcacgaaacgttgaccgcagcagcacagtacatgcgcgaacatgatatcggcgcattgccgatttgcggcgacgatgatcgtctgcacggtatgctgaccgaccgcgatatcgttatcaagggtctggccgcaggcttggacccgaacaccgcgaccgccggtgaactggcacgtgacagcatctattacgtcgacgcgaacgccagcattcaagagatgctgaacgtgatggaagagcatcaggtgcgtcgtgtcccggttatcagcgaacatcgtctggttggtatcgttaccgaagccgacatcgcacgtcacctgccggagcacgcgattgttcagttcgtgaaagcgatttgcagcccgatggcgttggcgtctcgtcaaaagggcgacacaaaatttattctaaatgca aagctt ttgctgggcctgagcaccattagcagcaaagcggatgacatcgactgggatgcgattgcgcagtgtgagagcggtggcaattgggcagcgaataccggcaatggcctgtacggcggtctgcagatctcccaggcgacgtgggacagcaatggtggcgtcggcagcccggctgccgcgtccccacaacaacagatcgaggtggcagataacattatgaaaacgcagggtccgggtgcttggccaaaatgctccagctgcagccagggtgacgcaccgctgggcagcctgacccacattctgacgttcctggcagcggaaaccggtggttgtagcggtagccgcgatgactga (SEQ ID NO: 71) MDFALLPPEVNSARMYTGPGAGSLLAAAGGWDSLAAELATTAEAYGSVLSGLAALHWRGPAAESMAVTAAPYIGWLYTTAEKTQQTAIQARAAALAFEQAYAMTLPPPVVAANRIQLLALIATNFFGQNTAAIAATEAQYAEMWAQDAAAMYGYATASAAAALLTPFSPPRQTTNPAGLTAQAAAVSQATDPLSLLIETVTQALQALTIPSFIPEDFTFLDAIFAGYATVGVTQDVESFVAGTIGAESNLGLLNVGDENPAEVTPGDFGIGELVSATSPGGGVSASGAGGAASVGNTVLASVGRANSIGQLSVPPSWAAPSTRPVSALSPAGLTTLPGTDVAEHGMPGVPGVPVAAGRASGVLPRYGVRLTVMAHPPAAGEFMIATTPvDREGATMITFRLRLPCRTILRVFSRNPLVRGTDRLEAVVMLLAVTVSLLTIPFAAAAGTAVQDSRSHVYAHQAQTRHPATATVIDHEGVIDSNTTATSAPPRTKITVPARWVVNGIERSGEVNAKPGTKSGDRVGIWVDSAGQLVDEPAPPARAIADAALAALGLWLSVAAVAGALLALTRAILIRVRNASWQHDIDSLFCTQRELMTTARDIMNAGVTCVGEHETLTAAAQYMREHDIGALPICGDDDRLHGMLTDRDIVIKGLAAGLDPNTATAGELARDSIYYVDANASIQEMLNVMEEHQVRRVPVISEHRLVGIVTEADIARHLPEHAIVQFVKAICSPMALASRQKGDTKFILNAKLLLGLSTISSKADDIDWDAIAQCESGGNWAANTGNGLYGGLQISQATWDSNGGVGSPAAASPQQQIEVADNIMKTQGPGAWPKCSSCSQGDAPLGSLTHILTFLAAETGGCSGSRDDKMK (SEQ ID NO: 72) Jatggattttgcgctgctgccgccggaagtgaacagcgcgcgcatgtataccggcccgggcgcgggcagcctgctggcggcggcgggcggctgggatagcctggcggcggaactggcgaccaccgcggaagcgtatggcagcgtgctgagcggcctggcggcgctgcattggcgcggcccggcggcggaaagcatggcggtgaccgcggcgccgtatattggctggctgtataccaccgcggaaaaaacccagcagaccgcgattcaggcgcgcgcggcggcgctggcgtttgaacaggcgtatgcgatgaccctgccgccgccggtggtggcggcgaaccgcattcagctgctggcgctgattgcgaccaacttttttggccagaacaccgcggcgattgcggcgaccgaagcgcagtatgcggaaatgtgggcgcaggatgcggcggcgatgtatggctatgcgaccgcgagcgcggcggcggcgctgctgaccccgtttagcccgccgcgccagaccaccaacccggcgggcctgaccgcgcaggcggcggcggtgagccaggcgaccgatccgctgagcctgctgattgaaaccgtgacccaggcgctgcaggcgctgaccattccgagctttattccggaagattttacctttctggatgcgatttttgcgggctatgcgaccgtgggcgtgacccaggatgtggaaagctttgtggcgggcaccattggcgcggaaagcaacctgggcctgctgaacgtgggcgatgaaaacccggcggaagtgaccccgggcgattttggcattggcgaactggtgagcgcgaccagcccgggcggcggcgtgagcgcgagcggcgcgggcggcgcggcgagcgtgggcaacaccgtgctggcgagcgtgggccgcgcgaacagcattggccagctgagcgtgccgccgagctgggcggcgccgagcacccgcccggtgagcgcgctgagcccggcgggcctgaccaccctgccgggcaccgatgtggcggaacatggcatgccgggcgtgccgggcgtgccggtggcggcgggccgcgcgagcggcgtgctgccgcgctatggcgtgcgcctgaccgtgatggcgcatccgccggcggcgggcgaatttatgacagagcagcagtggaatttcgcgggtatcgaggccgcggaagcgcaatccagggaaatgtcacgtccattcattccctccttgacgaggggaagcagtccctgaccaagctcgcagcggcctggggcggtagcggttcggaggcgtaccagggtgtccagcaaaaatgggacgccacggctaccgagctgaacaacgcgctgcagaacctggcgcggacgatcagcgaagccggtcaggcaatggcttcgaccgaaggcaacgtcactgggatgttcgca gaattc atgattgcgactacccgtgatcgtgagggcgcgaccatgatcacgttccgtctgcgtctgccgtgtcgcaccattttgcgcgtgttttcgcgtaacccgctggtccgcggtaccgaccgtctggaggccgttgtcatgctgctggcggttaccgtgagcctgctgacgatcccattcgcagcggcagctggcacggccgtccaagacagccgtagccatgtgtatgctcaccaggctcaaacccgtcacccggctactgccactgttatcgatcacgaaggcgtgattgactccaataccacggcaacctccgcaccgcctcgcaccaagattacggttcctgcgcgttgggtggtgaatggtattgaacgcagcggcgaagttaatgccaaaccgggtaccaaaagcggtgaccgtgtgggcatctgggtcgatagcgccggtcagctggtcgacgagccggcaccgccagcgcgtgcgatcgccgatgcggcgctggctgccctgggtctgtggctgagcgtggcagcggtcgccggtgcgttgctggcgctgacgcgcgcaattctgatccgcgttcgcaatgcgagctggcagcacgatattgatagcctgttttgcacccaacgt gagctc atgaccacggcgcgtgatatcatgaatgcgggtgtcacctgtgttggcgagcacgaaacgttgaccgcagcagcacagtacatgcgcgaacatgatatcggcgcattgccgatttgcggcgacgatgatcgtctgcacggtatgctgaccgaccgcgatatcgttatcaagggtctggccgcaggcttggacccgaacaccgcgaccgccggtgaactggcacgtgacagcatctattacgtcgacgcgaacgccagcattcaagagatgctgaacgtgatggaagagcatcaggtgcgtcgtgtcccggttatcagcgaacatcgtctggttggtatcgttaccgaagccgacatcgcacgtcacctgccggagcacgcgattgttcagttcgtgaaagcgatttgcagcccgatggcgttggcgtctcgtcaaaagggcgacacaaaatttattctaaatgca aagctt gcatgcaaaacggtgacgttgaccgtcgacggaaccgcgatgcgggtgaccacgatgaaatcgcgggtgatcgacatcgtcgaagagaacgggttctcagtcgacgaccgcgacgacctgtatcccgcggccggcgtgcaggtccatgacgccgacaccatcgtgctgcggcgtagccgtccgctgcagatctcgctggatggtcacgacgctaagcaggtgtggacgaccgcgtcgacggtggacgaggcgctggcccaactcgcgatgaccgacacggcgccggccgcggcttctcgcgccagccgcgtcccgctgtccgggatggcgctaccggtcgtcagcgccaagacggtgcagctcaacgacggcgggttggtgcgcacggtgcacttgccggcccccaatgtcgcggggctgctgagtgcggccggcgtgccgctgttgcaaagcgaccacgtggtgcccgccgcgacggccccgatcgtcgaaggcatgcagatccaggtgacccgcaatcggatcaagaaggtcaccgagcggctgccgctgccgccgaacgcgcgtcgtgtcgaggacccggagatgaacatgagccgggaggtcgtcgaagacccgggggttccggggacccaggatgtgacgttcgcggtagctgaggtcaacggcgtcgagaccggccgtttgcccgtcgccaacgtcgtggtgaccccggcccacgaagccgtggtgcgggtgggcaccaagcccggtaccgaggtgcccccggtgatcgacggaagcatctgggacgcgatcgccggctgtgaggccggtggcaactgggcgatcaacaccggcaacgggtattacggtggtgtgcagtttgaccagggcacctgggaggccaacggcgggctgcggtatgcaccccgcgctgacctcgccacccgcgaagagcagatcgccgttgccgaggtgacccgactgcgtcaaggttggggcgcctggccggtatgtgctgcacgagcgggtgcgcgctga (SEQ ID NO: 73) MDFALLPPEVNSARMYTGPGAGSLLAAAGGWDSLAAELATTAEAYGSVLSGLAALHWRGPAAESMAVTAAPYIGWLYTTAEKTQQTAIQARAAALAFEQAYAMTLPPPVVAANRIQLLALIATNFFGQNTAAIAATEAQYAEMWAQDAAAMYGYATASAAAALLTPFSPPRQTTNPAGLTAQAAAVSQATDPLSLLIETVTQALQALTIPSFIPEDFTFLDAIFAGYATVGVTQDVESFVAGTIGAESNLGLLNVGDENPAEVTPGDFGTGELVSATSPGGGVSASGAGGAASVGNTVLASVGRANSIGQLSVPPSWAAPSTRPVSALSPAGLTTLPGTDVAEHGMPGVPGVPVAAGRASGVLPRYGVRLTVMAHPPAAGEFMIATTRDREGATMITFRLRLPCRTILRVFSRNPLVRGTDRLEAVVMLLAVTVSLLTIPFAAAAGTAVQDSRSHVYAHQAQTRHPATATVIDHEGVIDSNTTATSAPPRTKITVPARWVVNGIERSGEVNAKPGTKSGDRVGIWVDSAGQLVDEPAPPARAIADAALAALGLWLSVAAVAGALLALTRAILIRVRNASWQHDIDSLFCTQRELMTTARDIMNAGVTCVGEHETLTAAAQYMREHDIGALPICGDDDRLHGMLTDRDIVIKGLAAGLDPNTATAGELARDSIYYVDANASIQEMLNVMEEHQVRRVPVISEHRLVGIVTEADIARHLPEHAIVQFVKAICSPMALASRQKGDTKFILNAKLACKTVTLTVDGTAMRVTTMKSRVIDIVEENGFSVDDRDDLYPAAGVQVHDADTIVLRRSRPLQISLDGHDAKQVWTTASTVDEALAQLAMTDTAPAAASRASRVPLSGMALPVVSAKTVQLNDGGLVRTVHLPAPNVAGLLSAAGVPLLQSDHVVPAATAPIVEGMQIQVTRNRIKKVTERLPLPPNARRVEDPEMNMSREVVEDPGVPGTQDVTFAVAEVNGVETGRLPVANVVVTPAHEAVVRVGTKPGTEVPPVIDGSIWDAIAGCEAGGNWAINTGNGYYGGVQFDQGTWEANGGLRYAPRADLATREEQIAVAEVTRLRQGWGAWPVCAARAGAR (SEQ ID NO: 74)

Any Mtb antigen, including any Mtb antigen within any of the fusionproteins described herein, can have an amino acid sequence that is 100%,or from 70% to 99.9%, identical to the particular amino acid sequencelisted in Tables 1-4. The amino acid sequence of any individual Mtbantigen, including any Mtb antigen within any of the fusion proteinsdescribed herein, can be at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical to the particular amino acidsequence listed in Tables 1-4. Identity or similarity with respect to anamino acid or nucleotide sequence is defined herein as the percentage ofamino acid residues (or nucleotide residues as the case may be) in theparticular Mtb antigen that are identical (i.e., same residue) with theamino acid or nucleotide sequence for the Mtb antigen shown in Tables1-4, after aligning the sequences and introducing gaps, if necessary, toachieve the maximum percent sequence identity. Percent sequence identitycan be determined by, for example, the Gap program (Wisconsin SequenceAnalysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, Madison Wis.), using default settings, whichuses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2,482-489). Any amino acid number calculated as a % identity can berounded up or down, as the case may be, to the closest whole number.

Any Mtb antigen, including any Mtb antigen within any of the fusionproteins described herein, can be fragments of the particular amino acidsequence listed in Tables 1-3. The amino acid sequence of any individualMtb antigen, including any Mtb antigen within any of the fusion proteinsdescribed herein, can be missing consecutive amino acids constituting atleast 20%, at least 15%, at least 10%, at least 5%, at least 4%, atleast 3%, at least 2%, or at least 1%, of the particular amino acidsequence listed in Tables 1-3. The omitted consecutive amino acids maybe from the C-terminus or N-terminus portion of the antigen.Alternately, the omitted consecutive amino acids may be from theinternal portion of the antigen, thus retaining at least its C-terminusand N-terminus amino acids of the antigen.

Any Mtb antigen, including any Mtb antigen within any of the fusionproteins described herein, can have one or more amino acid additions,deletions, or substitutions compared to the particular amino acidsequence listed in Tables 1-3. Any individual Mtb antigen, including anyMtb antigen within any of the fusion proteins described herein, can haveat least one, at least two, at least three, at least four, at leastfive, at least six, at least seven, at least eight, at least nine, atleast ten, at least eleven, or at least twelve amino acid additions,deletions, or substitutions compared to the particular amino acidsequence listed in Tables 1-3. The amino acid additions, deletions, orsubstitutions can take place at any amino acid position within the Mtbantigen.

Where a particular Mtb antigen, including any Mtb antigen within any ofthe fusion proteins described herein, comprises at least one or moresubstitutions, the substituted amino acid(s) can each be, independently,any naturally occurring amino acid or any non-naturally occurring aminoacid. Thus, a particular Mtb antigen may comprise one or more amino acidsubstitutions that are naturally occurring amino acids and/or one ormore amino acid substitutions that are non-naturally occurring aminoacids. Individual amino acid substitutions are selected from any one ofthe following: 1) the set of amino acids with nonpolar sidechains, forexample, Ala, Cys, Ile, Leu, Met, Phe, Pro, Val; 2) the set of aminoacids with negatively charged side chains, for example, Asp, Glu; 3) theset of amino acids with positively charged sidechains, for example, Arg,His, Lys; and 4) the set of amino acids with uncharged polar sidechains,for example, Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr, Trp, Tyr, towhich are added Cys, Gly, Met and Phe. Substitutions of a member of oneclass with another member of the same class are contemplated herein.Naturally occurring amino acids include, for example, alanine (Ala),arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys),glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His),isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met),phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr),tryptophan (Trp), tyrosine (Tyr), and valine (Val). Non-naturallyoccurring amino acids include, for example, norleucine, omithine,norvaline, homoserine, and other amino acid residue analogues such asthose described in Ellman et al., Meth. Enzym., 1991, 202, 301-336. Togenerate such non-naturally occurring amino acid residues, theprocedures of Noren et al., Science, 1989, 244, 182 and Ellman et al.,supra, can be used. Briefly, these procedures involve chemicallyactivating a suppressor tRNA with a non-naturally occurring amino acidresidue followed by in vitro transcription and translation of the RNA.

The Mtb antigens, including any Mtb antigen within any of the fusionproteins described herein, which are modified as described herein retaintheir ability to elicit an immune response against Mycobacteriumtuberculosis. That is, modification of a particular Mtb antigen,including any Mtb antigen within any of the fusion proteins describedherein, will still allow the resultant Mtb antigen, or fusion proteincomprising the same, to elicit an immune response against Mycobacteriumtuberculosis.

The present disclosure also provides nucleic acid molecules encoding anyof the fusion proteins described herein that comprise at least threeMycobacterium tuberculosis (Mtb) antigens. In some embodiments, thefusion protein comprises at least one acute Mtb antigen, at least onelatent Mtb antigen, and at least one resuscitation Mtb antigen. In someembodiments, the fusion protein comprises at least two latent Mtbantigens and at least one resuscitation Mtb antigen.

The nucleic acid molecules described herein and in Tables 1-4 arerepresentative. That is, the specific sequences recited in Tables 1-4are simply one example of a nucleic acid molecule that can encode aparticular Mtb antigen within a fusion protein. One skilled in the arthaving knowledge of the genetic code can routinely prepare and design aplethora of nucleic acid molecules encoding the same Mtb antigen. Thelength and nucleotide content of any particular nucleic acid molecule isdictated by the desired amino acid sequence of the encoded Mtb antigen.The nucleic acid molecule sequences shown in Tables 1-4 are DNA,although RNA nucleic acid molecules are also contemplated.

TABLE 5 Primer name Sequence 85B For NdeI ata gat cat atg ttt agc cgt cct ggc ctg c  (SEQ ID NO: 75) 85B Rev EcoRI nostoptta aga gaa ttc gcc cgc  acc cag aga gga t  (SEQ ID NO: 76)ESAT-6 For BamHI aac gtt gga tcc atg aca  gag cag cag tgg aa (SEQ ID NO: 77) ESAT-6 Rev EcoRI ns ata cta gaa ttc tgc gaa cat ccc agt gac gt  (SEQ ID NO: 78) 1733 For EcoRIaac tta gaa ttc atg att  gcg act acc cgt gat  (SEQ ID NO: 79)1733 In1 Rev Xma gat ata ccc ggg ggc ctc  cag acg gtc ggt (SEQ ID NO: 80) 1733 Out For Xma aac gaa ccc ggg gtc caa gac agc cgt agc c  (SEQ ID NO: 81) 1733 Out Rev Xbataa gta tct aga atc ggc  gat cgc acg cgc t  (SEQ ID NO: 82)1733 In2 For Xba ata gaa tct aga cgc gca  att ctg atc cgc gt (SEQ ID NO: 83) 1733 Rev ns SacI aga taa gag ctc acg ttg ggt gca aaa cag gc  (SEQ ID NO: 84) 2626 For SacIata gaa gag ctc atg acc  acg gcg cgt gat a  (SEQ ID NO: 85)2626 Rev HindIII ns taa aga aag ctt tgc att  tag aat aaa ttt tgt gtc (SEQ ID NO: 86) RpfD For HindIII taa cta aag ctt ttg ctg ggc ctg agc acc  (SEQ ID NO: 87) RpfD Rev XhoI stopatc taa ctc gag cta gtc  atc gcg gct acc gct  (SEQ ID NO: 88)ESAT6 For NdeI taa gat cat atg aca gag  cag cag tgg aat ttc (SEQ ID NO: 89) ESAT-6 Rev EcoRI ns ata cta gaa ttc tgc gaa cat ccc agt gac gt  (SEQ ID NO: 90)

The present disclosure also provides vectors encoding any of the Mtbantigens, including Mtb antigens within any of the fusion proteinsdescribed herein, including any of the modified versions describedherein. The vector can be capable of expressing an Mtb antigen in thecell of a mammal in a quantity effective to elicit an immune response inthe mammal. The vector can be recombinant. The vector can compriseheterologous nucleic acid encoding the antigen. The vector can be aplasmid. The vector can be useful for transfecting cells with nucleicacid encoding an Mtb antigen, which the transformed host cell iscultured and maintained under conditions wherein expression of theantigen takes place.

In some embodiments, coding sequences can be optimized for stability andhigh levels of expression. In some instances, codons are selected toreduce secondary structure formation of the RNA such as that formed dueto intramolecular bonding.

In some embodiments, the vectors can comprise regulatory elements forgene expression of the coding sequences of the nucleic acid. Theregulatory elements can be a promoter, an enhancer an initiation codon,a stop codon, or a polyadenylation signal. In some embodiments, thevector can comprise heterologous nucleic acid encoding an Mtb antigenand can further comprise an initiation codon, which is upstream of theantigen coding sequence, and a stop codon, which is downstream of theantigen coding sequence. The initiation and termination codon are inframe with the antigen coding sequence.

The vector can also comprise a promoter that is operably linked to theantigen coding sequence. The promoter operably linked to the Mtb antigencoding sequence can be a promoter from simian virus 40 (SV40), a mousemammary tumor virus (MMTV) promoter, a human immunodeficiency virus(HIV) promoter such as the bovine immunodeficiency virus (BIV) longterminal repeat (LTR) promoter, a Moloney virus promoter, an avianleukosis virus (ALV) promoter, a cytomegalovirus (CMV) promoter such asthe CMV immediate early promoter, Epstein Barr virus (EBV) promoter, ora Rous sarcoma virus (RSV) promoter, or the like. The promoter can alsobe a promoter from a human gene such as human actin, human myosin, humanhemoglobin, human muscle creatine, or human metalothionein. The promotercan also be a tissue specific promoter, such as a muscle or skinspecific promoter, natural or synthetic. Representative examples ofpromoters include the bacteriophage T7 promoter, bacteriophage T3promoter, SP6 promoter, lac operator-promoter, tac promoter,mycobacterial Hsp60 promoter, SV40 late promoter, SV40 early promoter,RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40 latepromoter and the CMV IE promoter.

The vector can also comprise a polyadenylation signal, which can bedownstream of the antigen coding sequence. The polyadenylation signalcan be a SV40 polyadenylation signal, LTR polyadenylation signal, CMVpolyadeylation signal, bovine growth hormone (bGH) polyadenylationsignal, human growth hormone (hGH) polyadenylation signal, or humanβ-globin polyadenylation signal. The SV40 polyadenylation signal can bea polyadenylation signal from a pCEP4 vector (Invitrogen, San Diego,Calif.).

The vector can also comprise an enhancer upstream of the consensusBoNT-A, BoNT-B, BoNT-E, and BoNT-F antigen sequences. The enhancer canbe necessary for DNA expression. The enhancer can be human actin, humanmyosin, human hemoglobin, human muscle creatine or a viral enhancer suchas one from CMV, HA, RSV or EBV. Polynucleotide function enhances aredescribed in U.S. Pat. Nos. 5,593,972, 5,962,428, and WO94/016737, thecontents of each are fully incorporated by reference.

The vector can also comprise a mammalian origin of replication in orderto maintain the vector extrachromosomally and produce multiple copies ofthe vector in a cell. The vector can be pVAX1, pCEP4 or pREP4 fromInvitrogen (San Diego, Calif.), which can comprise the Epstein Barrvirus origin of replication and nuclear antigen EBNA-1 coding region,which can produce high copy episomal replication without integration.The vector can be pVAX1 or a pVax1 variant with changes such as thevariant plasmid described herein. The variant pVax1 plasmid is a 2998basepair variant of the backbone vector plasmid pVAX1 (Invitrogen,Carlsbad Calif.). The CMV promoter is located at bases 137-724. The T7promoter/priming site is at bases 664-683. Multiple cloning sites are atbases 696-811. Bovine GH polyadenylation signal is at bases 829-1053.The Kanamycin resistance gene is at bases 1226-2020. The pUC origin isat bases 2320-2993.

The vector can also comprise a regulatory sequence, which can be wellsuited for gene expression in a mammalian or human cell into which thevector is administered. The consensus coding sequence can comprise acodon, which can allow more efficient transcription of the codingsequence in the host cell.

The vector can be pSE420 (Invitrogen, San Diego, Calif.) or pET28b (EMDMillipore, Billerca, Mass.), which can be used for protein production inEscherichia coli (E. coli). The vector can also be pYES2 (Invitrogen,San Diego, Calif.), which can be used for protein production inSaccharomyces cerevisiae strains of yeast. The vector can also be of theMAXBAC™ complete baculovirus expression system (Invitrogen, San Diego,Calif.), which can be used for protein production in insect cells. Thevector can also be pcDNA I or pcDNA3 (Invitrogen, San Diego, Calif.),which may be used for protein production in mammalian cells such asChinese hamster ovary (CHO) cells. The vector can be expression vectorsor systems to produce protein by routine techniques and readilyavailable starting materials including Sambrook et al., MolecularCloning and Laboratory Manual, Second Ed., Cold Spring Harbor (1989),which is incorporated fully by reference.

In some embodiments, the vector is a viral vector. Suitable viralvectors include, but are not limited to, an adenovirus vector, vacciniavirus vector, and paramyxovirus vector. Suitable adenovirus vectorsinclude, but are not limited to, adenovirus 4, adenovirus 5, chimpanzeeadenovirus 3, chimpanzee adenovirus 63, and chimpanzee adenovirus 68. Asuitable vaccinia virus vector includes, but is not limited to, modifiedvaccinia Ankara (MVA). Suitable paramyxovirus vectors include, but arenot limited to, modified parainfluenza virus (PIV2) and recombinanthuman parainfluenza virus (rHPIV2). In some embodiments, the vector ispresent within a composition comprising a pharmaceutically acceptablecarrier. One skilled in the art is readily familiar with numerousvectors, many of which are commercially available.

The present disclosure also provides host cells comprising any of thenucleic acid molecules or vectors disclosed herein. The host cells canbe used, for example, to express the Mtb antigens, or fragments ofthereof. The Mtb antigens, or fragments thereof, can also be expressedin cells in vivo. The host cell that is transformed (for example,transfected) to produce the Mtb antigens, or fragments of thereof can bean immortalised mammalian cell line, such as those of lymphoid origin(for example, a myeloma, hybridoma, trioma or quadroma cell line). Thehost cell can also include normal lymphoid cells, such as B-cells, thathave been immortalized by transformation with a virus (for example, theEpstein-Barr virus).

In some embodiments, the host cells include, but are not limited to:bacterial cells, such as E. coli, Caulobacter crescentus, Streptomycesspecies, and Salmonella typhimurium; yeast cells, such as Saccharomycescerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Pichiamethanolica; insect cell lines, such as those from Spodoptera frugiperda(for example, Sf9 and Sf21 cell lines, and expresSF™ cells (ProteinSciences Corp., Meriden, Conn., USA)), Drosophila S2 cells, andTrichoplusia in High Five® Cells (Invitrogen, Carlsbad, Calif., USA);and mammalian cells, such as COS1 and COS7 cells, Chinese hamster ovary(CHO) cells, NS0 myeloma cells, NIH 3T3 cells, 293 cells, Procell92S,perC6, HEPG2 cells, HeLa cells, L cells, HeLa, MDCK, HEK293, WI38,murine ES cell lines (for example, from strains 129/SV, C57/BL6, DBA-1,129/SVJ), K562, Jurkat cells, and BW5147. Other useful mammalian celllines are well known and readily available from the American TypeCulture Collection (“ATCC”) (Manassas, Va., USA) and the NationalInstitute of General Medical Sciences (NIGMS) Human Genetic CellRepository at the Coriell Cell Repositories (Camden, N.J., USA). In someembodiments, the cell is a recombinant BCG. These cell types are onlyrepresentative and are not meant to be an exhaustive list.

Among other considerations, some of which are described above, a hostcell strain may be chosen for its ability to process the expressed Mtbantigens, or fragment thereof, in the desired fashion.Post-translational modifications of the polypeptide include, but are notlimited to, glycosylation, acetylation, carboxylation, phosphorylation,lipidation, and acylation, and it is an aspect of the present disclosureto provide Mtb antigens thereof with one or more of thesepost-translational modifications.

In some embodiments, the recombinant BCG has been genetically engineeredto express a functional endosomalytic protein that is bioactive at pHvalues near neutrality (e.g. about pH 6-8 or about 6.5 to 7.5). Theendosomalytic protein is active within Mycobacteria-containingendosomes, which typically have an internal pH near neutrality. Theactivity of the endosomalytic protein produced by the rBCG results indisruption of the endosome, permitting the rBCG to escape from theendosome and into the cytoplasm of the cell. In some embodiments, theendosomalytic protein that is introduced into the rBCG by geneticengineering is Perfringolysin O (PfoA) from Clostridium perfringens or amutant thereof, such as PfoA_(G137Q), as described in WO 2007/058663,which is incorporated herein by reference in its entirety.

In some embodiments, the Mycobacteria are attenuated, as exemplified byBCG. However, those of skill in the art will recognize that otherattenuated and nonattenuated Mycobacteria exist which would also besuitable for use herein. Examples of additional types of Mycobacteriainclude, but are not limited to, M. tuberculosis strain CDC1551, M.tuberculosis strain Beijing, M. tuberculosis strain H37Ra (ATCC#:25177),M. tuberculosis strain H37Rv (ATCC#:25618), M. bovis (ATCC#:19211 and27291), M. fortuitum (ATCC#:15073), M. smegmatis (ATCC#:12051 and12549), M. intracellulare (ATCC#:35772 and 13209), M. kansasii(ATCC#:21982 and 35775) M. avium (ATCC#:19421 and 25291), M. gallinarum(ATCC#:19711), M. vaccae (ATCC#:15483 and 23024), M. leprae (ATCC#:), M.marinarum (ATCC#:11566 and 11567), and M. microtti (ATCC#:11152).

Examples of attenuated Mycobacterium strains include, but are notrestricted To, M. tuberculosis pantothenate auxotroph strain, M.tuberculosis rpoV mutant strain, M. tuberculosis leucine auxotrophstrain, BCG Danish strain (ATCC #35733), BCG Japanese strain (ATCC#35737), BCG Chicago strain (ATCC #27289), BCG Copenhagen strain (ATCC#: 27290), BCG Pasteur strain (ATCC #: 35734), BCG Glaxo strain (ATCC #:35741), BCG Connaught strain (ATCC #35745), BCG Montreal (ATCC #35746),BCG1331 strain, BCG Tokyo strain, BCG Moreau strain, BCG-Pasteur Aeras,and BCG Moscow strain.

In some embodiments, the cell comprising the one or more vector(s) ispresent within a composition comprising a pharmaceutically acceptablecarrier.

In some embodiments, the Mtb antigen, or fragment thereof, is labeledwith a detectable marker. Detectable markers include, but are notlimited to, radioactive isotopes (such as P³² and S³⁵), enzymes (such ashorseradish peroxidase, chloramphenicol acetyltransferase (CAT),β-galactosidase (β-gal), and the like), fluorochromes, chromophores,colloidal gold, dyes, and biotin. The labeled Mtb antigens, or fragmentsthereof, can be used to carry out diagnostic procedures in a variety ofcell or tissue types. For imaging procedures, in vitro or in vivo, theMtb antigens can be labeled with additional agents, such as NMRcontrasting agents, X-ray contrasting agents, or quantum dots. Methodsfor attaching a detectable agent to polypeptides are known in the art.The Mtb antigens can also be attached to an insoluble support (such as abead, a glass or plastic slide, or the like).

In some embodiments, the Mtb antigens, or fragment thereof, can beconjugated to a therapeutic agent including, but not limited to,radioisotopes (such as ¹¹¹In or ⁹⁰Y), toxins (such as tetanus toxoid orricin), toxoids, and chemotherapeutic agents.

In some embodiments, the Mtb antigens, or fragments thereof, can beconjugated to an imaging agent. Imaging agents include, for example, alabeling moiety (such as biotin, fluorescent moieties, radioactivemoieties, histidine tag or other peptide tags) for easy isolation ordetection.

The present disclosure also provides compositions comprising at leastthree Mycobacterium tuberculosis (Mtb) antigens, wherein the compositioncomprises: at least one acute Mtb antigen, at least one latent Mtbantigen, and at least one resuscitation Mtb antigen; or at least twolatent Mtb antigens, and at least one resuscitation Mtb antigen. In someembodiments, the at least three Mtb antigens are not present in a fusionprotein. In some embodiments, the at least three Mtb antigens are in theform of a protein and not nucleic acid molecules encoding the Mtbantigens.

In some embodiments, the acute Mtb antigen is Ag85B, ESAT6, MPT64,PPE15, PPE51, or Rv3615c. In some embodiments, the latent Mtb antigen isRv1733c, Rv2626c, Rv3407, or Rv2628c. In some embodiments, the firstand/or second transmembrane region of Rv1733c is deleted (Rv1733cΔTM).In some embodiments, the resuscitation Mtb antigen is RpfB, RpfD, orRpfE. In some embodiments, the composition comprises at least four Mtbantigens. In some embodiments, the composition comprises: ESAT6,Rv1733c, Rv2626c, and RpfD Mtb antigens; ESAT6, Rv1733c, Rv2626c, andRpfB Mtb antigens; RpfB, ESAT6, Rv1733c, and Rv2626c Mtb antigens;Ag85B, ESAT6, Rv1733c, Rv2626c, and RpfD Mtb antigens; Ag85B, ESAT6,Rv1733c, Rv2626c, and RpfB Mtb antigens; PPE51, Rv1733c, Rv2628c, andRpfD Mtb antigens; PPE51, Rv1733c, Rv2628c, and RpfB Mtb antigens;Rv3407, Rv1733c, Rv2626c, and RpfB Mtb antigens; or Rv3407, Rv1733c,Rv2626c, and RpfD Mtb antigens. In some embodiments, the compositionfurther comprises a pharmaceutically acceptable carrier.

The present disclosure also provides compositions comprising at leastthree Mycobacterium tuberculosis (Mtb) antigens, wherein the compositioncomprises: at least one acute Mtb antigen, at least one latent Mtbantigen, and at least one resuscitation Mtb antigen; or at least twolatent Mtb antigens, and at least one resuscitation Mtb antigen; whereinthe composition comprises at least one nucleic acid molecule encoding atleast one of the Mtb antigens. In some embodiments, the compositioncomprises one Mtb antigen in protein form and one or two nucleic acidmolecules encoding two Mtb antigens. In some embodiments, thecomposition comprises two Mtb antigens in protein form, optionally as afusion protein, and one nucleic acid molecule encoding one Mtb antigen.Thus, the present composition is a mixture of a protein Mtb antigen(s)and nucleic acid molecule(s) encoding an Mtb antigen(s).

In some embodiments, at least two Mtb antigens are encoded by one ormore nucleic acid molecules within one or more vectors. In someembodiments, the one or more vectors is one or more viral vectors. Insome embodiments, the one or more viral vectors are any one or more ofadenovirus vector, vaccinia virus vector, or paramyxovirus vector. Insome embodiments, the adenovirus vector is adenovirus 4, adenovirus 5,chimpanzee adenovirus 3, chimpanzee adenovirus 63, or chimpanzeeadenovirus 68. In some embodiments, the one or more vaccinia virusvector is modified vaccinia Ankara (MVA). In some embodiments, the oneor more paramyxovirus vectors are any one or more of modifiedparainfluenza virus (PIV2 or PIV3) or recombinant human parainfluenzavirus (rHPIV2). In some embodiments, the at least two Mtb antigens areencoded by a single nucleic acid molecule within the same expressionvector as a fusion protein.

In some embodiments, the acute Mtb antigen is Ag85B, ESAT6, MPT64,PPE15, PPE51, or Rv3615c. In some embodiments, the latent Mtb antigen isRv1733c, Rv2626c, Rv3407, or Rv2628c. In some embodiments, the firstand/or second transmembrane region of Rv1733c is deleted. In someembodiments, the resuscitation Mtb antigen is RpfB, RpfD, or RpfE. Insome embodiments, the composition comprises at least four Mtb antigens.In some embodiments, the composition comprises: ESAT6, Rv1733c, Rv2626c,and RpfD Mtb antigens; ESAT6, Rv1733c, Rv2626c, and RpfB Mtb antigens;RpfB, ESAT6, Rv1733c, and Rv2626c Mtb antigens; Ag85B, ESAT6, Rv1733c,Rv2626c, and RpfD Mtb antigens; Ag85B, ESAT6, Rv1733c, Rv2626c, and RpfBMtb antigens; PPE51, Rv1733c, Rv2628c, and RpfD Mtb antigens; PPE51,Rv1733c, Rv2628c, and RpfB Mtb antigens; Rv3407, Rv1733c, Rv2626c, andRpfB Mtb antigens; or Rv3407, Rv1733c, Rv2626c, and RpfD Mtb antigens;wherein any two or more Mtb antigens can be within a fusion protein. Insome embodiments, the composition comprises at least four Mtb antigens.In some embodiments, the composition further comprises apharmaceutically acceptable carrier.

In some embodiments, where a rBCG is used as the vehicle to deliver theMtb antigens, or fusion proteins, or nucleic acids and or vectorscomprising or encoding the same, expression of all or part of the Dos Rregulon is not up-regulated in the rBCG. In some embodiments, one ormore of the following Dos R regulon antigens are not up-regulated in therBCG: Rv1738, Rv2623, Rv2031c, Rv2032, Rv2626c, Rv2005c, Rv3127,Rv1733c, Rv1996, Rv2628c, Rv0079, Rv3130c, Rv3131, Rv1813c, Rv2006,Rv2029c, Rv2627c, Rv2030c, Rv3132c, and Rv2629. In some embodiments, therBCG does not comprise up-regulation of: 1) one or more Mtb antigens,including “classical” Mtb antigens such as 85A, 85B and TB 10.4; and 2)at least one Mtb resuscitation antigen selected from Rv0867c, Rv1009,Rv1884c, Rv2389c, Rv2450c, Rv0288, Rv1009, Rv0685, Rv0824c, Rv1349,Rv2744c, Rv3347c, Rv1130, and Rv1169c. In some embodiments, the rBCGdoes not include the expression of the following combinations: classicalantigens Rv1886c, Rv3804c; resuscitation antigens Rv0867c, Rv1884c,Rv2389c; and Mtb-specific antigen Rv3407. In some embodiments, the rBCGdoes not include the expression of the following combination: Rv3804c,Rv1886c, and Rv3407, or in addition with Rv3133c, and with thecombination of Rv0867c, Rv1884c, and Rv2389c. In some embodiments, therBCG does not include the expression of the following combination:TB10.4, Ag85B, Ag85A, and Rv3407. In some embodiments, the cell is not arBCG.

The present disclosure also provides compositions comprising any one ormore of the fusion proteins, Mtb antigens, nucleic acid moleculesencoding Mtb antigens, including fusion proteins thereof, cells, and/orvectors and a pharmaceutically acceptable carrier. Compositions include,for example, pharmaceutical compositions. A pharmaceutically acceptablecarrier refers to at least one component of a pharmaceutical preparationthat is normally used for administration of active ingredients. As such,a carrier can contain any pharmaceutical excipient used in the art andany form of vehicle for administration. Carriers include, but are notlimited to, phosphate buffered saline, physiological saline, water,citrate/sucrose/Tween formulations and emulsions such as, for example,oil/water emulsions.

The compositions can also include an active therapeutic agent and avariety of other pharmaceutically acceptable components. See Remington'sPharmaceutical Science (15th ed., Mack Publishing Company, Easton, Pa.(1980)). The desired form depends on the intended mode of administrationand therapeutic application. The compositions can also include,depending on the formulation desired, pharmaceutically acceptable,non-toxic carriers or diluents, which are defined as vehicles commonlyused to formulate pharmaceutical compositions for animal or humanadministration. The diluent is selected so as not to affect thebiological activity of the combination. Examples of such diluentsinclude, but are not limited to, distilled water, physiologicalphosphate-buffered saline, Ringer's solutions, dextrose solution, andHank's solution. In addition, the pharmaceutical composition orformulation may also include other carriers, adjuvants, or nontoxic,nontherapeutic, nonimmunogenic stabilizers and the like.

Solid formulations of the compositions for oral administration cancontain suitable carriers or excipients, such as corn starch, gelatin,lactose, acacia, sucrose, microcrystalline cellulose, kaolin, mannitol,dicalcium phosphate, calcium carbonate, sodium chloride, or alginicacid. Disintegrators that can be used include, without limitation,microcrystalline cellulose, corn starch, sodium starch glycolate, andalginic acid. Tablet binders that can be used include acacia,methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone(Povidone™), hydroxypropyl methylcellulose, sucrose, starch, andethylcellulose. Lubricants that can be used include magnesium stearates,stearic acid, silicone fluid, talc, waxes, oils, and colloidal silica.Additional excipients include, for example, colorants, taste-maskingagents, solubility aids, suspension agents, compressing agents, entericcoatings, sustained release aids, and the like.

In some embodiments, the compositions can be administered in the form ofa depot injection or implant preparation, which can be formulated insuch a manner as to permit a sustained release. An exemplary compositioncomprises any one or more of the compositions described hereinformulated in aqueous buffer.

In some embodiments, liquid formulations of a pharmaceutical compositionfor oral administration prepared in water or other aqueous vehicles cancontain various suspending agents such as methylcellulose, alginates,tragacanth, pectin, kelgin, carrageenan, acacia, polyvinylpyrrolidone,and polyvinyl alcohol. Liquid formulations of pharmaceuticalcompositions can also include solutions, emulsions, syrups and elixirscontaining, together with the active compound(s), wetting agents,sweeteners, and coloring and flavoring agents. Various liquid and powderformulations of the pharmaceutical compositions can be prepared byconventional methods for inhalation into the lungs of the mammal to betreated.

In some embodiments, liquid formulations of a pharmaceutical compositionfor injection can comprise various carriers such as vegetable oils,dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate,isopropyl myristate, ethanol, polyols such as, for example, glycerol,propylene glycol, liquid polyethylene glycol, and the like. In someembodiments, the composition includes a citrate/sucrose/tween carrier.For intravenous injections, water soluble versions of the compositionscan be administered by the drip method, whereby a pharmaceuticalformulation containing the antifungal agent and a physiologicallyacceptable excipient is infused. Physiologically acceptable excipientscan include, for example, 5% dextrose, 0.9% saline, Ringer's solution orother suitable excipients. A suitable insoluble form of the compositioncan be prepared and administered as a suspension in an aqueous base or apharmaceutically acceptable oil base, such as an ester of a long chainfatty acid such as, for example, ethyl oleate.

The compositions can be, for example, injectable solutions, aqueoussuspensions or solutions, non-aqueous suspensions or solutions, solidand liquid oral formulations, salves, gels, ointments, intradermalpatches, creams, lotions, tablets, capsules, sustained releaseformulations, and the like. In some embodiments, for topicalapplications, the pharmaceutical compositions can be formulated in asuitable ointment. In some embodiments, a topical semi-solid ointmentformulation typically comprises a concentration of the active ingredientfrom about 1 to 20%, or from 5 to 10%, in a carrier, such as apharmaceutical cream base. Some examples of formulations of acomposition for topical use include, but are not limited to, drops,tinctures, lotions, creams, solutions, and ointments containing theactive ingredient and various supports and vehicles.

Typically, compositions are prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid vehicles prior to injection can also be prepared.The preparation also can be emulsified or encapsulated in liposomes ormicroparticles such as polylactide, polyglycolide, or copolymer forenhanced adjuvant effect (see Langer, Science, 1990, 249, 1527 andHanes, Advanced Drug Delivery Reviews, 1997, 28, 97). A sterileinjectable preparation such as, for example, a sterile injectableaqueous or oleaginous suspension can also be prepared. This suspensionmay be formulated according to techniques known in the art usingsuitable dispersing, wetting, and suspending agents. In someembodiments, the pharmaceutical composition can be delivered in amicroencapsulation device so as to reduce or prevent a host immuneresponse against the protein.

Effective doses of the compositions of the present disclosure, for thetreatment of a condition vary depending upon many different factors,including means of administration, target site, physiological state ofthe subject, whether the subject is human or an animal, othermedications administered, and whether treatment is prophylactic ortherapeutic. Usually, the subject is a human but non-human mammalsincluding transgenic mammals can also be treated.

In some embodiments, the compositions can be administered to a subjectby injection intravenously, subcutaneously, intraperitoneally,intramuscularly, intramedullarily, intraventricularly, intraepidurally,intraarterially, intravascularly, intraarticularly, intrasynovially,intrasternally, intrathecally, intrahepatically, intraspinally,intratumorly, intracranially, enteral, intrapulmonary, transmucosal,intrauterine, sublingual, or locally at sites of inflammation or tumorgrowth by using standard methods. Alternately, the compositions can beadministered to a subject by routes including oral, nasal, ophthalmic,rectal, or topical. The most typical route of administration isintravascular, subcutaneous, or intramuscular, although other routes canbe effective. In some embodiments, compositions are administered as asustained release composition or device, such as a Medipad™ device. Thecomposition can also be administered via the respiratory tract, forexample, using a dry powder inhalation device, nebulizer, or a metereddose inhaler. The composition can also be administered by traditionalsyringes, needleless injection devices, “microprojectile bombardmentgone guns,” or other physical methods such as electroporation (“EP”),“hydrodynamic method”, or ultrasound.

In some embodiments, the composition can be administered to a subject bysustained release administration, by such means as depot injections oferodible implants directly applied during surgery or by implantation ofan infusion pump or a biocompatible sustained release implant into thesubject. Alternately, the composition can be administered to a subjectby injectable depot routes of administration, such as by using 1-, 3-,or 6-month depot injectable or biodegradable materials and methods, orby applying to the skin of the subject a transdermal patch containingthe composition, and leaving the patch in contact with the subject'sskin, generally for 1 to 5 hours per patch.

In some embodiments, the compositions comprise about 1 nanogram to about10 mg of nucleic acid. In some embodiments, the compositionscomprise: 1) at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95 or 100 nanograms, or at least 1, 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105,110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175,180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245,250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315,320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385,390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455,460, 465, 470, 475, 480, 485, 490, 495, 500, 605, 610, 615, 620, 625,630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695,700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765,770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825, 830, 835,840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895. 900, 905,910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975,980, 985, 990, 995 or 1000 micrograms, or at least 1.5, 2, 2.5, 3, 3.5,4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 mg or more; and 2)up to and including 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95 or 100 nanograms, or up to and including 1, 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170,175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240,245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310,315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380,385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450,455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 605, 610, 615, 620,625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690,695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760,765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825, 830,835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895. 900,905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970,975, 980, 985, 990, 995, or 1000 micrograms, or up to and including 1.5,2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 mg.

In some embodiments, the compositions comprise about 5 nanograms toabout 10 mg of nucleic acid molecule. In some embodiments, thecompositions comprise about 25 nanograms to about 5 mg of nucleic acidmolecule. In some embodiments, the compositions contain about 50nanograms to about 1 mg of nucleic acid molecule. In some embodiments,the compositions contain about 0.1 to about 500 micrograms of nucleicacid molecule. In some embodiments, the compositions contain about 1 toabout 350 micrograms of nucleic acid molecule. In some embodiments, thecompositions contain about 5 to about 250 micrograms of nucleic acidmolecule. In some embodiments, the compositions contain about to about200 micrograms of nucleic acid molecule. In some embodiments, thecompositions contain about 15 to about 150 micrograms of nucleic acidmolecule. In some embodiments, the compositions contain about 20 toabout 100 micrograms of nucleic acid molecule. In some embodiments, thecompositions contain about 25 to about 75 micrograms of nucleic acidmolecule. In some embodiments, the compositions contain about 30 toabout 50 micrograms of nucleic acid molecule. In some embodiments, thecompositions contain about 35 to about 40 micrograms of nucleic acidmolecule. In some embodiments, the compositions contain about 100 toabout 200 micrograms of nucleic acid molecule. In some embodiments, thecompositions comprise about 10 to about 100 micrograms of nucleic acidmolecule. In some embodiments, the compositions comprise about 20 toabout 80 micrograms of nucleic acid molecule. In some embodiments, thecompositions comprise about 25 to about 60 micrograms of nucleic acidmolecule. In some embodiments, the compositions comprise about 30nanograms to about 50 micrograms of nucleic acid molecule. In someembodiments, the compositions comprise about 35 nanograms to about 45micrograms of nucleic acid molecule. In some embodiments, thecompositions contain about 0.1 to about 500 micrograms of nucleic acidmolecule. In some embodiments, the compositions contain about 1 to about350 micrograms of nucleic acid molecule. In some embodiments, thecompositions contain about 25 to about 250 micrograms of nucleic acidmolecule. In some embodiments, the compositions contain about 100 toabout 200 micrograms of nucleic acid molecule.

The compositions can be formulated according to the mode ofadministration to be used. In cases where compositions are injectablepharmaceutical compositions, they are sterile, pyrogen free andparticulate free. An isotonic formulation can be used. Generally,additives for isotonicity can include sodium chloride, dextrose,mannitol, sorbitol and lactose. In some cases, isotonic solutions suchas phosphate buffered saline are suitable. Stabilizers include gelatinand albumin. In some embodiments, a vasoconstriction agent is added tothe formulation.

The compositions can further comprise a pharmaceutically acceptableexcipient. The pharmaceutically acceptable excipient can be functionalmolecules as vehicles, adjuvants, carriers, or diluents. Thepharmaceutically acceptable excipient can be a transfection facilitatingagent, which can include surface active agents, such asimmune-stimulating complexes (ISCOMS), Freund's incomplete adjuvant, LPSanalog including monophosphoryl lipid A, muramyl peptides, quinoneanalogs, vesicles such as squalene and squalane, hyaluronic acid,lipids, liposomes, calcium ions, viral proteins, polyanions,polycations, or nanoparticles, or other known transfection facilitatingagents. The transfection facilitating agent is a polyanion, polycation,including poly-L-glutamate (LGS), or lipid. The transfectionfacilitating agent is poly-L-glutamate, and more suitably, thepoly-L-glutamate is present in the composition at a concentration lessthan 6 mg/ml. The transfection facilitating agent can also includesurface active agents such as immune-stimulating complexes (ISCOMS),Freunds incomplete adjuvant, LPS analog including monophosphoryl lipidA, muramyl peptides, quinone analogs and vesicles such as squalene andsqualane, and hyaluronic acid can also be used administered inconjunction with the genetic construct. In some embodiments, the plasmidcompositions can also include a transfection facilitating agent such aslipids, liposomes, including lecithin liposomes or other liposomes knownin the art, as a DNA-liposome mixture (see for example WO9324640),calcium ions, viral proteins, polyanions, polycations, or nanoparticles,or other known transfection facilitating agents. In some embodiments,the transfection facilitating agent is a polyanion, polycation,including poly-L-glutamate (LGS), or lipid. Concentration of thetransfection agent in the composition is less than 4 mg/ml, less than 2mg/ml, less than 1 mg/ml, less than 0.750 mg/ml, less than 0.500 mg/ml,less than 0.250 mg/ml, less than 0.100 mg/ml, less than 0.050 mg/ml, orless than 0.010 mg/ml.

The pharmaceutically acceptable excipient may be an adjuvant. Theadjuvant may be other genes that are expressed in alternative plasmid orare delivered as proteins in combination with the plasmid above. Theadjuvant may be selected from the group consisting of: α-interferon(IFN-α), β-interferon (IFN-β), γ-interferon, platelet derived growthfactor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF),cutaneous T cell-attracting chemokine (CTACK), epithelialthymus-expressed chemokine (TECK), mucosae-associated epithelialchemokine (MEC), IL-12, IL-15, MHC, CD80, CD86 including IL-15 havingthe signal sequence deleted and optionally including the signal peptidefrom IgE. The adjuvant may be IL-12, IL-15, IL-28, CTACK, TECK, plateletderived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growthfactor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, or acombination thereof.

Other genes which may be useful adjuvants include those encoding: MCP-1,MIP-1a, MIP-1p, IL-8, L-selectin, P-selectin, E-selectin, CD34,GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, p150.95, PECAM, ICAM-1, ICAM-2,ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40,CD40L, vascular growth factor, fibroblast growth factor, IL-7, nervegrowth factor, vascular endothelial growth factor, Fas, TNF receptor,Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5,KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-1, Ap-1,Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, Inactive NIK, SAP K, SAP-1,JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec,TRAILrecDRCS, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND,NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP 1, TAP2 andfunctional fragments thereof.

The plasmid compositions can further comprise a genetic vaccinefacilitator agent as described in U.S. Ser. No. 021,579 filed Apr. 1,1994, which is fully incorporated by reference.

The present disclosure also provides kits comprising any of the Mtbantigens, fragments thereof, fusion proteins, nucleic acid molecules,vectors, or cells, described herein. The kit can include, for example,container(s), package(s) or dispenser(s) along with labels andinstructions for administration or use.

The present disclosure also provides methods of eliciting an immuneresponse against Mycobacterium tuberculosis in a mammal comprisingadministering to the mammal an immunologically sufficient amount of oneor more fusion proteins comprising at least three Mycobacteriumtuberculosis (Mtb) antigens, wherein at least one fusion proteincomprises: at least one acute Mtb antigen, at least one latent Mtbantigen, and at least one resuscitation Mtb antigen; or at least twolatent Mtb antigens, and at least one resuscitation Mtb antigen. Any ofthe fusion proteins described herein can be administered. In someembodiments, the fusion protein comprises at least four Mtb antigens.

The present disclosure also provides methods of eliciting an immuneresponse against Mycobacterium tuberculosis in a mammal comprisingadministering to the mammal an immunologically sufficient amount of acomposition comprising at least three Mycobacterium tuberculosis (Mtb)antigens, wherein the composition comprises: at least one acute Mtbantigen, at least one latent Mtb antigen, and at least one resuscitationMtb antigen; or at least two latent Mtb antigens, and at least oneresuscitation Mtb antigen; and a pharmaceutically acceptable carrier.Any of the compositions comprising three or more Mtb antigens can beadministered. In some embodiments, the composition comprises at leastfour Mtb antigens.

The present disclosure also provides methods of eliciting an immuneresponse against Mycobacterium tuberculosis in a mammal comprisingadministering to the mammal an immunologically sufficient amount of acomposition comprising at least three Mycobacterium tuberculosis (Mtb)antigens, wherein the composition comprises: at least one acute Mtbantigen, at least one latent Mtb antigen, and at least one resuscitationMtb antigen; or at least two latent Mtb antigens, and at least oneresuscitation Mtb antigen; and a pharmaceutically acceptable carrier;wherein the composition comprises at least one nucleic acid moleculeencoding at least one of the Mtb antigens. Any of the compositionscomprising a mixture of one or more Mtb antigen proteins and one of morenucleic acid molecules encoding one or more Mtb antigens describedherein can be administered.

The fusion proteins and compositions described herein can be used totreat or prevent tuberculosis. In some embodiments, the method comprisesadministering to a human a therapeutically- orprophylactically-effective amount of any of the fusion proteins orcompositions described herein such that the tuberculosis infection isdiminished or prevented.

In some embodiments, the subject being treated will have been previouslydiagnosed as having tuberculosis. Such subjects will, thus, have beendiagnosed as being in need of such treatment. Alternately, the treatmentmay be intended to prevent a tuberculosis infection in a subject thatdoes not yet have tuberculosis or to a subject that is travelling to anarea where tuberculosis is prevalent.

Treatment of a subject suffering from tuberculosis can be monitoredusing standard methods. Some methods entail determining a baselinevalue, for example, of an antibody level or profile in a subject, beforeadministering a dosage of agent, and comparing this with a value for theprofile or level after treatment. A significant increase such as, forexample, greater than the typical margin of experimental error in repeatmeasurements of the same sample, expressed as one standard deviationfrom the mean of such measurements in value of the level or profilesignals a positive treatment outcome (i.e., that administration of theagent has achieved a desired response). If the value for immune responsedoes not change significantly, or decreases, a negative treatmentoutcome is indicated.

In other embodiments, a control value such as a mean and standarddeviation, of level or profile is determined for a control population.Typically the individuals in the control population have not receivedprior treatment. Measured values of the level or profile in a subjectafter administering a therapeutic agent are then compared with thecontrol value. A significant increase relative to the control value,such as greater than one standard deviation from the mean, signals apositive or sufficient treatment outcome. A lack of significant increaseor a decrease signals a negative or insufficient treatment outcome.Administration of the therapeutic is generally continued while the levelis increasing relative to the control value. As before, attainment of aplateau relative to control values is an indicator that theadministration of treatment can be discontinued or reduced in dosageand/or frequency.

In other embodiments, a control value of the level or profile, such as amean and standard deviation, is determined from a control population ofindividuals who have undergone treatment with a therapeutic agent andwhose levels or profiles have plateaued in response to treatment.Measured values of levels or profiles in a subject are compared with thecontrol value. If the measured level in a subject is not significantlydifferent, such as by more than one standard deviation, from the controlvalue, treatment can be discontinued. If the level in a subject issignificantly below the control value, continued administration of agentis warranted. If the level in the subject persists below the controlvalue, then a change in treatment may be indicated.

In other embodiments, a subject who is not presently receiving treatmentbut has undergone a previous course of treatment is monitored forantibody levels or profiles to determine whether a resumption oftreatment is required. The measured level or profile in the subject canbe compared with a value previously achieved in the subject after aprevious course of treatment. A significant decrease relative to theprevious measurement, such as greater than a typical margin of error inrepeat measurements of the same sample, is an indication that treatmentcan be resumed. Alternately, the value measured in a subject can becompared with a control value (mean plus standard deviation) determinedin a population of subjects after undergoing a course of treatment.Alternately, the measured value in a subject can be compared with acontrol value in populations of prophylactically treated subjects whoremain free of symptoms of disease, or populations of therapeuticallytreated subjects who show amelioration of disease characteristics. Inall of these cases, a significant decrease relative to the controllevel, such as more than a standard deviation, is an indicator thattreatment should be resumed in a subject.

In some methods, a baseline measurement of antibody to a given antigenin the subject is made before administration, a second measurement ismade soon thereafter to determine the peak antibody level, and one ormore further measurements are made at intervals to monitor decay ofantibody levels. When the level of antibody has declined to baseline ora predetermined percentage of the peak less baseline, such as 50%, 25%or 10%, administration of a further dosage of antigen is administered.In some embodiments, peak or subsequent measured levels less backgroundare compared with reference levels previously determined to constitute abeneficial prophylactic or therapeutic treatment regime in othersubjects. If the measured antibody level is significantly less than areference level, such as less than the mean minus one standard deviationof the reference value in population of subjects benefiting fromtreatment, administration of an additional dosage of antigen isindicated.

In some embodiments, the subject(s) that can be treated by theabove-described methods is an animal, such as a mammal, including, butare not limited to, humans, non-human primates, rodents (including rats,mice, hamsters and guinea pigs) cow, horse, sheep, goat, pig, dog andcat. In most instances, the mammal is a human.

The present disclosure also provides fusion proteins for use in thepreparation of a medicament for treating or preventing a Mycobacteriumtuberculosis infection, wherein the fusion protein comprises at leastthree Mycobacterium tuberculosis (Mtb) antigens, and wherein the fusionprotein comprises: at least one acute Mtb antigen, at least one latentMtb antigen, and at least one resuscitation Mtb antigen; or at least twolatent Mtb antigens, and at least one resuscitation Mtb antigen.

The present disclosure also provides fusion proteins for use in treatingor preventing a Mycobacterium tuberculosis infection, wherein the fusionprotein comprises at least three Mycobacterium tuberculosis (Mtb)antigens, and wherein the fusion protein comprises: at least one acuteMtb antigen, at least one latent Mtb antigen, and at least oneresuscitation Mtb antigen; or at least two latent Mtb antigens, and atleast one resuscitation Mtb antigen.

The present disclosure also provides uses of a fusion protein in thepreparation of a medicament for treating or preventing a Mycobacteriumtuberculosis infection, wherein the fusion protein comprises at leastthree Mycobacterium tuberculosis (Mtb) antigens, and wherein the fusionprotein comprises: at least one acute Mtb antigen, at least one latentMtb antigen, and at least one resuscitation Mtb antigen; or at least twolatent Mtb antigens, and at least one resuscitation Mtb antigen.

The present disclosure also provides uses of a fusion protein intreating or preventing a Mycobacterium tuberculosis infection, whereinthe fusion protein comprises at least three Mycobacterium tuberculosis(Mtb) antigens, and wherein the fusion protein comprises: at least oneacute Mtb antigen, at least one latent Mtb antigen, and at least oneresuscitation Mtb antigen; or at least two latent Mtb antigens, and atleast one resuscitation Mtb antigen.

The present disclosure also provides compositions for use in thepreparation of a medicament for treating or preventing a Mycobacteriumtuberculosis infection, wherein the composition comprises at least threeMycobacterium tuberculosis (Mtb) antigens, and wherein the compositioncomprises: at least one acute Mtb antigen, at least one latent Mtbantigen, and at least one resuscitation Mtb antigen; or at least twolatent Mtb antigens, and at least one resuscitation Mtb antigen; and apharmaceutically acceptable carrier.

The present disclosure also provides compositions for use in treating orpreventing a Mycobacterium tuberculosis infection, wherein thecomposition comprises at least three Mycobacterium tuberculosis (Mtb)antigens, and wherein the composition comprises: at least one acute Mtbantigen, at least one latent Mtb antigen, and at least one resuscitationMtb antigen; or at least two latent Mtb antigens, and at least oneresuscitation Mtb antigen; and a pharmaceutically acceptable carrier.

The present disclosure also provides uses of a composition in thepreparation of a medicament for treating or preventing a Mycobacteriumtuberculosis infection, wherein the composition comprises at least threeMycobacterium tuberculosis (Mtb) antigens, and wherein the compositioncomprises: at least one acute Mtb antigen, at least one latent Mtbantigen, and at least one resuscitation Mtb antigen; or at least twolatent Mtb antigens, and at least one resuscitation Mtb antigen; and apharmaceutically acceptable carrier.

The present disclosure also provides uses of a composition in treatingor preventing a Mycobacterium tuberculosis infection, wherein thecomposition comprises at least three Mycobacterium tuberculosis (Mtb)antigens, and wherein the composition comprises: at least one acute Mtbantigen, at least one latent Mtb antigen, and at least one resuscitationMtb antigen; or at least two latent Mtb antigens, and at least oneresuscitation Mtb antigen; and a pharmaceutically acceptable carrier.

The present disclosure also provides compositions for use in thepreparation of a medicament for treating or preventing a Mycobacteriumtuberculosis infection, wherein the composition comprises at least threeMycobacterium tuberculosis (Mtb) antigens, and wherein the compositioncomprises: at least one acute Mtb antigen, at least one latent Mtbantigen, and at least one resuscitation Mtb antigen; or at least twolatent Mtb antigens, and at least one resuscitation Mtb antigen; and apharmaceutically acceptable carrier; wherein the composition comprisesat least one nucleic acid molecule encoding at least one of the Mtbantigens.

The present disclosure also provides compositions for use in treating orpreventing a Mycobacterium tuberculosis infection, wherein thecomposition comprises at least three Mycobacterium tuberculosis (Mtb)antigens, and wherein the composition comprises: at least one acute Mtbantigen, at least one latent Mtb antigen, and at least one resuscitationMtb antigen; or at least two latent Mtb antigens, and at least oneresuscitation Mtb antigen; and a pharmaceutically acceptable carrier;wherein the composition comprises at least one nucleic acid moleculeencoding at least one of the Mtb antigens.

The present disclosure also provides uses of a composition in thepreparation of a medicament for treating or preventing a Mycobacteriumtuberculosis infection, wherein the composition comprises at least threeMycobacterium tuberculosis (Mtb) antigens, and wherein the compositioncomprises: at least one acute Mtb antigen, at least one latent Mtbantigen, and at least one resuscitation Mtb antigen; or at least twolatent Mtb antigens, and at least one resuscitation Mtb antigen; and apharmaceutically acceptable carrier; wherein the composition comprisesat least one nucleic acid molecule encoding at least one of the Mtbantigens.

The present disclosure also provides uses of a composition in treatingor preventing a Mycobacterium tuberculosis infection, wherein thecomposition comprises at least three Mycobacterium tuberculosis (Mtb)antigens, and wherein the composition comprises: at least one acute Mtbantigen, at least one latent Mtb antigen, and at least one resuscitationMtb antigen; or at least two latent Mtb antigens, and at least oneresuscitation Mtb antigen; and a pharmaceutically acceptable carrier;wherein the composition comprises at least one nucleic acid moleculeencoding at least one of the Mtb antigens.

The present disclosure also provides any of the fusion proteinsdescribed herein, or any of the compositions described herein, or any ofthe cells described herein, or any of the vectors described herein, orany of the methods described herein, or any of the uses describedherein, substantially as described with reference to the accompanyingexamples and/or figures.

In order that the subject matter disclosed herein may be moreefficiently understood, examples are provided below. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting the claimed subject matter in anymanner. Throughout these examples, molecular cloning reactions, andother standard recombinant DNA techniques, were carried out according tomethods described in Maniatis et al., Molecular Cloning—A LaboratoryManual, 2nd ed., Cold Spring Harbor Press (1989), using commerciallyavailable reagents, except where otherwise noted.

EXAMPLES Example 1 Construction of the Antigen Cassette as the BasicTool for Subsequent Platform Insertion

The 5 antigen cassette (Construct D), which was human codon optimized,was synthesized commercially by Aldevron and cloned into pVAX-1. For usein MVA vectors, antigen 85B was synthesized with its native leadersequence. For viral vectors other than MVA, antigen 85B was replacedwith genes either containing or not containing the leader sequence, thisbeing achieved using the unique EcoRI and XmaI nuclease targetsequences. To clone the 5 antigen cassette into adenoviral or CMVvectors, primers with homology arms were used to PCR amplify thecassette, and this PCR product was recombined into the appropriateregion of the BAC.

For recombinant protein expression of Construct D, the 85B, Rv1733,Rv2626, and RpfD genes were synthesized by DNA2.0 and codon optimizedfor E. coli expression. Antigen 85B and RpfD were synthesized withoutthe native leader sequences. Each gene was PCR amplified from therespective DNA2.0 vector with appropriate restriction sites added andcloned into pET28b sequentially. ESAT-6 was PCR amplified from H37RvDNA.

More specifically, the genes encoding the protein antigens were PCRamplified using the primers in Table 5 and cloned into the pET28b vector(Novagen) via the indicated restriction enzyme sites. ESAT6 was PCRamplified from Mtb and first cloned into the pET23b vector (Novagen). Itwas subsequently PCR amplified and cloned into pET28b. The genes forantigen 85B, Rv1733c, Rv2626c, and RpfD were all synthesized with theircodons optimized for expression in E. coli (DNA2.0). Antigen Ag85B andrpfD were synthesized without the bases encoding the N-terminal signalsequence, and rpfB was PCR amplified from Mtb without the N-terminalsignal sequence. The codon optimized genes were PCR amplified and clonedinto pET28b creating N-terminal 6xHis-tagged fusion proteins. The geneswere cloned with no spacer sequences, only the restriction enzyme sitesbetween each gene. To remove the 2 transmembrane regions of Rv1733c, itwas PCR amplified in 3 pieces which were ligated together.

In another embodiment the 4 Ag and 5 Ag proteins were constructed withwild type Rv1733c including the transmembrane regions. The pET28bconstructs were cloned in E. coli cloning strains, screened byrestriction digest and sequenced to verify each construct.

Example 2 Construction of Recombinant BCG (rBCG) Strains

rBCG strains over-expressing antigens involved with active infection,latency, and resuscitation were constructed. The genes of interest werefirst cloned in a plasmid which allows for their insertion in thechromosome of BCG at the attB integration site (pJFINT-RIAR). Since thisplasmid has three different cloning sites, the 5 genes were not fusedtogether but rather split into three groups. Ag85B was fused to ESAT-6(Ag85B-ESAT6; SEQ ID NO:91 (nucleotide) and SEQ ID NO:92 (amino acid)with Ag85B signal sequence; SEQ ID NO:93 (nucleotide) and SEQ ID NO:94(amino acid) with 19 kDa lipoprotein signal sequence) and placed in thefirst cloning site, Rv1733c was cloned by itself, and Rv2626c was fusedwith RpfD (Rv2626c-RpfD; SEQ ID NO:95 and SEQ ID NO:96 with Ag85B signalsequence; SEQ ID NO:97 and SEQ ID NO:98 with 19 kDa lipoprotein signalsequence) then placed into the third site to create the followingconstruct: Ag85B-ESAT6+Rv1733c+Rv2626c-RpfD. Each insert was placedunder the control of the Hsp60 promoter and a signal sequence was addedto both fusions, except Rv1733c since it is already a mycobacterialmembrane protein. Constructs with two different signal sequences weremade; the Ag85B signal sequence which allows for secretion of thefusions, and the 19 kDa lipoprotein signal sequence which anchors theminto the membrane to examine which one would allow better expressionand/or immunogenicity of the antigens. Both replicating andnon-replicating versions of those rBCG strains were constructed. Themaps of the plasmids that were used to construct the rBCG strains areshown in FIG. 1A (fusions with the Ag85B signal sequence) and 1B(fusions with the 19 kDa signal sequence).

After the rBCG strains were constructed by integration of the shuttleplasmids in the chromosome of BCG SSI, BCG SSIΔPanCD or other BCGstrains, cell lysates and supernatants were prepared to evaluate therelative expression of the different antigens as well as theirlocalization in the rBCG cells. Western blot data using a monoclonalantibody against ESAT-6 as a probe showed the following: the BCG SSIcontrol, which does not have the gene for ESAT-6, showed no reactivitywith the monoclonal antibody in either the cell lysate or the culturesupernatant; for rBCG expressing the Ag85B-ESAT6 fusion linked to the 19kDa signal sequence, low levels of the fusion were detected, but only inthe cell lysates and not in the culture supernatant of both thereplicating and non-replicating rBCG strains, showing that this signalsequence does not result in secretion of the fusion, as expected; therBCG strain expressing the Ag85B-ESAT6 fusion linked to the Ag85B signalsequence showed a very high level of expression both in the cell lysateand the culture supernatant confirming that this signal sequence doesresult in the secretion of this fusion (data not shown).

Following the antigen expression studies, a preliminary immunogenicityexperiment was carried out in C57/BL6 mice comparing the BCG SSI controland the rBCG strains expressing the fusions with the two differentsignal sequences. Mice were immunized once sc with either 10⁵ or 10⁶CFUs of the different BCG strains and were sacrificed 6 weeks later. Thesplenocytes were stimulated for 72 hours with recombinant Ag85B orESAT-6 proteins and the levels of antigen specific IFNγ released in theculture supernatants were measured using an ELISA assay. The followingresults were obtained with the Ag85B-ESAT6 fusions (see, FIG. 2): theBCG SSI control given at the lowest dose showed background levels ofIFNγ, similar to what was obtained with the naïve mice, whereas the micegiven the highest dose showed higher levels of Ag85B specific IFNγ, butno ESAT6 specific response which was expected since the BCG SSI controldoes not have the gene for ESAT-6; in contrast, the rBCG strainexpressing the Ag85B-ESAT6 fusion linked to the Ag85B signal sequencegave a much stronger Ag85B specific response at both doses, but anESAT-6 response above background only at the higher dose; similarresults were obtained with the rBCG strain expressing the fusion linkedto the 19 kDa signal sequence, but only at the higher dose, which is notsurprising considering the expression levels were much lower in thatstrain (data not shown).

DNA sequence of the Ag85B-ESAT6 fusion with the Ag85B signal sequence:

(SEQ ID NO: 91) atgacagacgtgagccgaaagattcgagcttggggacgccgattgatgatcggcacggcagcggctgtagtccttccgggcctggtggggcttgccggcggagcggcaaccgcgggcgcgttctcccggccggggctgccggtcgagtacctgcaggtgccgtcgccgtcgatgggccgcgacatcaaggttcagttccagagcggtgggaacaactcacctgcggtttatctgctcgacggcctgcgcgcccaagacgactacaacggctgggatatcaacaccccggcgttcgagtggtactaccagtcgggactgtcgatagtcatgccggtcggcgggcagtccagcttctacagcgactggtacagcccggcctgcggtaaggctggctgccagacttacaagtgggaaaccttcctgaccagcgagctgccgcaatggttgtccgccaacagggccgtgaagcccaccggcagcgctgcaatcggcttgtcgatggccggctcgtcggcaatgatcttggccgcctaccacccccagcagttcatctacgccggctcgctgtcggccctgctggacccctctcaggggatggggcctagcctgatcggcctcgcgatgggtgacgccggcggttacaaggccgcagacatgtggggtccctcgagtgacccggcatgggagcgcaacgaccctacgcagcagatccccaagctggtcgcaaacaacacccggctatgggtttattgcgggaacggcaccccgaacgagttgggcggtgccaacatacccgccgagttcttggagaacttcgttcgtagcagcaacctgaagttccaggatgcgtacaacgccgcgggcgggcacaacgccgtgttcaacttcccgcccaacggcacgcacagctgggagtactggggcgctcagctcaacgccatgaagggtgacctgcagagttcgttaggcgccggcatgacagagcagcagtggaatttcgcgggtatcgaggccgcggcaagcgcaatccagggaaatgtcacgtccattcattccctccttgacgaggggaagcagtccctgaccaagctcgcagcggcctggggcggtagcggttcggaggcgtaccagggtgtccagcaaaaatgggacgccacggctaccgagctgaacaacgcgctgcagaacctggcgcggacgatcagcgaagccggtcaggcaatggcttcgaccgaaggcaacgtcactgggatgttcgcatga.Amino acid sequence of the Ag85B-ESAT6 fusion with the Ag85B signalsequence:

(SEQ ID NO: 92) MTDVSRKIRAWGRRLMIGTAAAVVLPGLVGLAGGAATAGAFSRPGLPVEYLQVPSPSMGRDIKVQFQSGGNNSPAVYLLDGLRAQDDYNGWDINTPAFEWYYQSGLSIVMPVGGQSSFYSDWYSPACGKAGCQTYKWETFLTSELPQWLSANRAVKPTGSAAIGLSMAGSSAMILAAYHPQQFIYAGSLSALLDPSQGMGPSLIGLAMGDAGGYKAADMWGPSSDPAWERNDPTQQIPKLVANNTRLWVYCGNGTPNELGGANIPAEFLENFVRSSNLKFQDAYNAAGGHNAVFNFPPNGTHSWEYWGAQLNAMKGDLQSSLGAGMTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWDATATELNNALQNLARTISEAGQAMASTEGNVTGMFA.DNA sequence of the Ag85B-ESAT6 fusion with the 19 kDa signal sequence:

(SEQ ID NO: 93) atgaagcgtggactgacggtcgcggtagccggagccgccattctggtcgcaggtattccggatgttcaagcaacaagtcgactacaggaagcggtgagaccacgaccgcggcaggtaccacggcaagccccggccggccggggctgccggtcgagtacctgcaggtgccgtcgccgtcgatgggccgcgacatcaaggttcagttccagagcggtgggaacaactcacctgcggtttatctgacgacggcctgcgcgcccaagacgactacaacggctgggatatcaacaccccggcgttcgagtggtactaccagtcgggactgtcgatagtcatgccggtcggcgggcagtccagatctacagcgactggtacagcccggcctgcggtaaggctggctgccagacttacaagtgggaaaccttcctgaccagcgagagccgcaatggttgtccgccaacagggccgtgaagcccaccggcagcgctgcaatcggcttgtcgatggccggctcgtcggcaatgatcttggccgcctaccacccccagcagttcatctacgccggctcgctgtcggccagaggacccactcaggggatggggcctagcctgatcggcctcgcgatgggtgacgccggcggttacaaggccgcagacatgtggggtccacgagtgacccggcatgggagcgcaacgaccctacgcagcagatccccaagaggtcgcaaacaacacccggctatgggtttattgcgggaacggcaccccgaacgagttgggcggtgccaacatacccgccgagttcttggagaacttcgttcgtagcagcaacctgaagttccaggatgcgtacaacgccgcgggcgggcacaacgccgtgttcaacttcccgcccaacggcacgcacagctgggagtactggggcgctcagctcaacgccatgaagggtgacctgcagagttcgttaggcgccggcatgacagagcagcagtggaatttcgcgggtatcgaggccgcggcaagcgcaatccagggaaatgtcacgtccattcattccctccttgacgaggggaagcagtccctgaccaagctcgcagcggcctggggcggtagcggttcggaggcgtaccagggtgtccagcaaaaatgggacgccacggctaccgagctgaacaacgcgctgcagaacctggcgcggacgatcagcgaagccggtcaggcaatggatcgaccgaaggcaacgtcactgggatgttcgcatga.Amino acid sequence of the Ag85B-ESAT6 fusion with the 19 kDa signalsequence:

(SEQ ID NO: 94) MKRGLTVAVAGAAILVAGLSGCSSNKSTTGSGETTTAAGTTASPGRPGLPVEYLQVPSPSMGRDIKVQFQSGGNNSPAVYLLDGLRAQDDYNGWDINTPAFEWYYQSGLSIVMPVGGQSSFYSDWYSPACGKAGCQTYKWETFLTSELPQWLSANRAVKPTGSAAIGLSMAGSSAMILAAYHPQQFIYAGSLSALLDPSQGMGPSLIGLAMGDAGGYKAADMWGPSSDPAWERNDPTQQIPKLVANNTRLWVYCGNGTPNELGGANIPAEFLENFVRSSNLKFQDAYNAAGGHNAVFNFPPNGTHSWEYWGAQLNAMKGDLQSSLGAGMTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWDATATELNNALQNLARTISEAGQAMASTEGNVTGMFA.DNA sequence of Rv2626c-RpfD fusion with the Ag85B signal sequence:

(SEQ ID NO: 95) atgacagacgtgagccgaaagattcgagcttggggacgccgattgatgatcggcacggcagcggctgtagtccttccgggcctggtggggcttgccggcggagcggcaaccgcgggcgcgttctccatgaccaccgcacgcgacatcatgaacgcaggtgtgacctgtgttggcgaacacgagacgctaaccgctgccgctcaatacatgcgtgagcacgacatcggcgcgttgccgatctgcggggacgacgaccggctgcacggcatgctcaccgaccgcgacattgtgatcaaaggcctggctgcgggcctagacccgaataccgccacggctggcgagttggcccgggacagcatctactacgtcgatgcgaacgcaagcatccaggagatgctcaacgtcatggaagaacatcaggtccgccgtgttccggtcatctcagagcaccgcttggtcggaatcgtcaccgaagccgacatcgcccgacacctgcccgagcacgccattgtgcagttcgtcaaggcaatctgctcgcccatggccctcgccagcatgacaccgggtttgcttactactgcgggtgctggccgaccacgtgacaggtgcgccaggatcgtatgcacggtgttcatcgaaaccgccgttgtcgcgaccatgtttgtcgcgttgttgggtctgtccaccatcagctcgaaagccgacgacatcgattgggacgccatcgcgcaatgcgaatccggcggcaattgggcggccaacaccggtaacgggttatacggtggtctgcagatcagccaggcgacgtgggattccaacggtggtgtcgggtcgccggcggccgcgagtccccagcaacagatcgaggtcgcagacaacattatgaaaacccaaggcccgggtgcgtggccgaaatgtagttcttgtagtcagggagacgcaccgctgggctcgctcacccacatcctgacgttcctcgcggccgagactggaggttgttcggggagcagggacgattag.Amino acid sequence of the Rv2626c-RpfD fusion with the Ag85B signalsequence:

(SEQ ID NO: 96) MTDVSRKIRAWGRRLMIGTAAAVVLPGLVGLAGGAATAGAFSMTTARDIMNAGVTCVGEHETLTAAAQYMREHDIGALPICGDDDRLHGMLTDRDIVIKGLAAGLDPNTATAGELARDSIYYVDANASIQEMLNVMEEHQVRRVPVISEHRLVGIVTEADIARHLPEHAIVQFVKAICSPMALASMTPGLLTTAGAGRPRDRCARIVCTVFIETAVVATMFVALLGLSTISSKADDIDWDAIAQCESGGNWAANTGNGLYGGLQISQATWDSNGGVGSPAAASPQQQIEVADNIMKTQGPGAWPKCSSCSQGDAPLGSLTHILTFLAAE TGGCSGSRDD.DNA sequence of the Rv2626c-RpfD fusion with the 19 kDa signal sequence:

(SEQ ID NO: 97) Atgaagcgtggactgacggtcgcggtagccggagccgccattctggtcgcaggtattccggatgttcaagcaacaagtcgactacaggaagcggtgagaccacgaccgcggcaggtaccacggcaagccccggcatgaccaccgcacgcgacatcatgaacgcaggtgtgacctgtgttggcgaacacgagacgctaaccgctgccgctcaatacatgcgtgagcacgacatcggcgcgttgccgatctgcggggacgacgaccggctgcacggcatgctcaccgaccgcgacattgtgatcaaaggcctggctgcgggcctagacccgaataccgccacggctggcgagttggcccgggacagcatctactacgtcgatgcgaacgcaagcatccaggagatgctcaacgtcatggaagaacatcaggtccgccgtgttccggtcatctcagagcaccgcttggtcggaatcgtcaccgaagccgacatcgcccgacacctgcccgagcacgccattgtgcagttcgtcaaggcaatctgctcgcccatggccctcgccagcatgacaccgggtttgcttactactgcgggtgctggccgaccacgtgacaggtgcgccaggatcgtatgcacggtgttcatcgaaaccgccgttgtcgcgaccatgtttgtcgcgttgttgggtagtccaccatcagctcgaaagccgacgacatcgattgggacgccatcgcgcaatgcgaatccggcggcaattgggcggccaacaccggtaacgggttatacggtggtctgcagatcagccaggcgacgtgggattccaacggtggtgtcgggtcgccggcggccgcgagtccccagcaacagatcgaggtcgcagacaacattatgaaaacccaaggcccgggtgcgtggccgaaatgtagttcttgtagtcagggagacgcaccgctgggctcgctcacccacatcctgacgttcctcgcggccgagactggaggttgttcggggagcagggacgattag.Amino acid sequence of the Rv2626c-RpfD fusion with the 19 kDa signalsequence:

(SEQ ID NO: 98) MKRGLTVAVAGAAILVAGLSGCSSNKSTTGSGETTTAAGTTASPGMTTARDIMNAGVTCVGEHETLTAAAQYMREHDIGALPICGDDDRLHGMLTDRDIVIKGLAAGLDPNTATAGELARDSIYYVDANASIQEMLNVMEEHQVRRVPVISEHRLVGIVTEADIARHLPEHAIVQFVKAICSPMALASMTPGLLTTAGAGRPRDRCARIVCTVFIETAVVATMFVALLGLSTISSKADDIDWDAIAQCESGGNWAANTGNGLYGGLQISQATWDSNGGVGSPAAASPQQQIEVADNIMKTQGPGAWPKCSSCSQGDAPLGSLTHILTFL AAETGGCSGSRDD.

Example 3 Cloning and Overexpression of Fusion Proteins of the Cassetteand Variants

Preparation of the Antigen Cassette and its Variants as Fusion ProteinRequired a Modified Strategy Outline Below:

Cloning:Multiple recombinant fusion proteins were created, of which twoare exemplified here: one with four Mtb antigens(ESAT6-Rv1733c-Rv2626c-RpfD), and one with five antigens(Ag85B-ESAT6-Rv1733c-Rv2626c-RpfD). The genes encoding the proteinantigens were PCR amplified using the primers in Table 5 and cloned intothe pET28b vector (Novagen) via the indicated restriction enzyme sites.ESAT6 was PCR amplified from Mtb and first cloned into the pET23b vector(Novagen). It was subsequently PCR amplified and cloned into pET28b. Thegenes for antigen 85B, Rv1733c, Rv2626c, and RpfD were all synthesizedwith their codons optimized for expression in E. coli (DNA2.0). Ag85B,and rpfD were synthesized without the bases encoding the N-terminalsignal sequence, and rpfB was PCR amplified from Mtb without theN-terminal signal sequence. The codon optimized genes were PCR amplifiedand cloned into pET28b creating N-terminal 6xHis-tagged fusion proteins.The genes were cloned with no spacer sequences, only the restrictionenzyme sites between each gene. To remove the 2 transmembrane regions ofRv1733c, it was PCR amplified in 3 pieces which were ligated together.In another embodiment the 4 Ag and 5 Ag proteins were constructed withwild type Rv1733c including the transmembrane regions. The pET28bconstructs were cloned in E. coli cloning strains, screened byrestriction digest and sequenced to verify each construct. The DNA andamino acid sequences of the 4 Ag and 5 Ag fusions were prepared withoutthe transmembrane regions of Rv1733c. Later versions of these fusionsreplaced RpfD with RpfB in the 4 Ag fusion, with RpfB placed either atthe 5′ or the 3′ end of the fusion.

Expression:The plasmids encoding Construct D and its variant fusionproteins were transformed into E. coli T7 express (NEB) or E. coli BL21DE3 (Novagen). Multiple colonies of each fusion construct were pickedand grown overnight shaking at 37° C. in Tryptic Soy Broth (TSB)(Sigma). Overnight cultures were diluted 1:100 in TSB and grown shakingat 37° C. to OD600=0.6. Cultures were induced with 1 mM IPTG and grownshaking at 37° C. for 3 hours. Induced and uninduced aliquots of eachculture were run on 4-12% Bis/Tris SDS-PAGE gels to verify induction ofthe fusion proteins. Colonies expressing each of the fusion proteinswere frozen in TSB+20% glycerol at −80° C. as research stocks.

Purification of Fusion Proteins

10 ml cultures were inoculated from glycerol stocks of the BE1726D andits variant fusion constructs and grown overnight shaking at 37° C. Theovernight cultures were diluted 1:100 in 250 ml TSB and grown shaking at37° C. to OD600=0.6. Cultures were induced with 1 mM IPTG and grownshaking at 37° C. for 3 hours. An aliquot of the induced sample was runon a 4-12% Bis/Tris SDS-PAGE gel to confirm induction of the protein.The induced culture was centrifuged at 6,000×g for 10 m and pellets werefrozen at −80° C. Pellets were thawed and resuspended in 10 ml BPERbuffer (Thermo Scientific), and an aliquot was taken for testing(lysate). Lysozyme (20 u/ml) and DNase I (25 U/ml) was added to helpcomplete cell lysis. The lysed cells were centrifuged at 12,000×g for 10minutes and the supernatant was collected (soluble fraction). Theinsoluble pellet was resuspended in 10 ml BPER buffer and a 100 μlaliquot was removed (insoluble fraction). The cells in the resuspendedpellet were diluted with 10 ml 10% BPER buffer and the suspension wascentrifuged at 12,000×g for 10 minutes. The supernatant was discardedand the pellet was washed again with 10 ml 10% BPER buffer 3 more times.The lysate, soluble and insoluble fractions and washes were run on a4-12% Bis/Tris SDS-PAGE gel to confirm expression and determine thesubcellular localization of the protein. The fusion proteins were foundlocalized to the insoluble pellet in inclusion bodies. The insolublepellet was resuspended in 10 ml denaturing binding buffer (DBB) (8 Murea, 92 mM Na₂HPO₄, 7 mM NaH₂PO₄, 10 mM Tris) pH 7.8. The inclusionbodies were lysed by sonication, and the lysate was cleared of debris bycentrifugation at 12,000×g for 20 minutes.

Proteins with the transmembrane regions of Rv1733c deleted were purifiedby column purification. Five (5) ml of HisPur Cobalt resin (ThermoScientific) was equilibrated with DBB and incubated with 5 ml of clearedlysate. The mixture was rocked at room temperature for 90 minutes. Thelysate/resin mixture was then loaded on a 30 ml column and washed with25 volumes of denaturing wash buffer (8 M urea, 25 mM Na₂HPO₄, 75 mMNaH₂PO₄, 10 mM Tris, 12 mM sodium deoxycholate, pH 7.8). His-taggedprotein was eluted from the Co+ column by eluting with elution buffer (8M urea, 10 mM Tris, 5% glycerol) pH 8.0 with 50, 100, 350, 500, and 1000mM imidazole. Eluted proteins were run on a 4-12% Bis/Tris SDS-PAGE geland clean fractions were dialyzed stepwise from 8M urea, 10 mM Tris, 5%glycerol to 10 mM Tris, 5% glycerol. Dialyzed protein was analyzed bySDS-PAGE for purity, western blot for the presence of each antigen, andwas assayed for the presence of residual endotoxin. Pure samples with<0.25 U endotoxin/ml were aliquoted and frozen at −80° C.

Proteins which have wild type Rv1733c were purified using an AKTApurifier (GE Healthcare). After the inclusion bodies were separated byBPER washes as above, the insoluble pellet was resuspended in 10 mldenaturing binding buffer (DBB)+20 mM imidazole. The inclusion bodieswere lysed by sonication, and the lysate was cleared of debris bycentrifugation at 12,000×g for 20 minutes. Five (5) ml of Ni SepharoseHigh Performance (GE Healthcare) resin was equilibrated with DBB andincubated with 10 ml of cleared lysate. The mixture was rocked at roomtemperature for 2 hours. The mixture was then loaded on the AKTApurifier. All the lines used on AKTA purifier were equilibrated withDBB+20 mM imidazole, denaturing wash buffer (8 M urea, 25 mM Na₂HPO₄, 75mM NaH₂PO₄, 10 mM Tris, 12 mM sodium deoxycholate, 20 mM imidazole) pH7.8, or elution buffer (8 M urea, 10 mM Tris, 5% glycerol, 20 mMimidazole), as needed. Proteins were eluted by gradient elution (elutionbuffer 1: 8 M urea, 10 mM Tris, 5% glycerol, 20 mM imidazole, run from100% to 0; elution buffer 2: 8 M urea, 10 mM Tris, 5% glycerol, 500 mMimidazole, fun from 0 to 100%) and fractions were collected. Thepositive fractions were run on a 4-12% Bis-Tris SDS-PAGE gel and weredialyzed stepwise from 8 M urea, 10 mM Tris, 5% glycerol to 10 mM Tris,5% glycerol. Dialyzed protein was analyzed by SDS-PAGE for purity,western blot for the presence of each antigen, and was assayed for thepresence of residual endotoxin. Pure samples with <0.25 U endotoxin/mlwere aliquoted and frozen at −80° C.

The foregoing describes the purification of the 4 Ag and 5 Ag proteinsthat were expressed with 6xHis tags. The proteins can also be expressedwithout tags. The untagged proteins can be purified by combiningchromatographic methods including ion exchange and size exclusionchromatography and filtration methods such as tangential flow.

Example 4 Immunogenicity of the 5 Ag and 4 Ag Fusion Proteins in Mice

The 5 Ag fusion protein was tested for immunogenicity in CB6F1 mice,adjuvanted with a synthetic poly I:C TLR3 agonist. Multiple otheradjuvants, such as TLR4 agonists, were tested and shown to beimmunogenic, and thus the embodiment is independent of the adjuvantused, and applicable to many classes of adjuvants. Mice were immunizedsubcutaneously twice, two weeks apart, with 1 or 10 μg of adjuvantedfusion protein. Two (2) weeks after the second immunization, the micewere sacrificed and splenocytes were isolated. The splenocytes wereincubated with recombinant protein antigens for in vitro stimulation andrecombinant protein or overlapping peptides for ELISpot analysis. The 5Ag fusion protein induced significant IFN-γ responses to each antigenthat were measurable by both in vitro stimulation and ELISpot (see,FIGS. 3A and 3B). The response to Ag85B, the most immunogenic and firstantigen of the fusion protein, was much stronger to the responses to theother antigens.

The 4 Ag and 5 Ag proteins were then both tested for immunogenicity inCB6F1 mice, adjuvanted with a synthetic MPL TLR4 agonist. Mice wereimmunized subcutaneously twice, two weeks apart, with 3 μg adjuvantedfusion protein. Splenocytes were isolated for in vitro stimulation andELISpot. Splenocytes were stimulated with individual antigens or fusionproteins Immunization with either the 4 Ag or 5 Ag fusion proteinsinduced IFN-γ responses to all antigens. Responses to ESAT6, Rv1733c,Rv2626c, and RpfD were all higher in the 4 Ag fusion protein, whichlacks Ag85B, than the 5 Ag fusion protein (see, FIGS. 4A and 4B).

Immunogenicity studies were also performed on the fusion proteins withthe wild-type Rv1733c (85B-ESAT6-Rv1733cwt-Rv2626c-RpfD andESAT6-Rv1733cwt-Rv2626c-RpfD) and the 4 Ag fusions where RpfD wasreplaced by RpfB (ESAT6-Rv1733c-Rv2626c-RpfB andRpfB-ESAT6-Rv1733c-Rv2626c). These studies compared the immunogenicityof fusion proteins containing the modified Rv1733c with that of fusionscontaining the wild-type Rv1733c, and also the immunogenicity of fusionproteins containing RpfD with that of fusions containing RpfB. Thestudies showed that while replacing the modified Rv1733c with thewild-type 1733c did not affect overall immunogenicity, RpfB issignificantly more immunogenic than RpfD in these fusions (see, FIGS. 5Aand 5B).

Example 5 Ongoing Protective Efficacy

The 5 Ag and 4 Ag fusion proteins (BE1726D, E1726D) were used in a primeboost protection experiment in mice. Mice were primed with BCG SSI andrecombinant BCG SSI overexpressing the 5 Mtb antigens that make up the 5Ag fusion protein. Six (6) and 8 weeks later, the mice were boosted witheither the 5 Ag or the 4 Ag protein plus a poly I:C adjuvant. Four (4)weeks after the second boost mice received an aerosol Mtb challenge of50-100 CFU. Mice were then sacrificed at 4 and 12 weeks post challengeto determine viable bacteria in the lungs (see, FIG. 6A) and spleen(see, FIG. 6B). This experiment determined whether the large response toAg85B is more protective in mice than the more broad response to theother 4 antigens in the 4 Ag fusion protein.

Various modifications of the described subject matter, in addition tothose described herein, will be apparent to those skilled in the artfrom the foregoing description. Such modifications are also intended tofall within the scope of the appended claims. Each reference (including,but not limited to, journal articles, U.S. and non-U.S. patents, patentapplication publications, international patent application publications,gene bank accession numbers, and the like) cited in the presentapplication is incorporated herein by reference in its entirety.

What is claimed is:
 1. A fusion protein consisting of four or fiveantigens, wherein the four or five antigens are Mycobacteriumtuberculosis antigens, and wherein the fusion protein comprises an aminoacid sequence that is at least 98% identical to an amino acid sequenceof: ESAT6-Rv1733c-Rv2626c-RpfD (SEQ ID NO:53 or SEQ ID NO:54);ESAT6-Rv1733c-Rv2626c-RpfB (SEQ ID NO:56); RpfB-ESAT6-Rv1733c-Rv2626c(SEQ ID NO:58); Ag85B-ESAT6-Rv1733c-Rv2626c-RpfD (SEQ ID NO:61 or SEQ IDNO:62); Ag85B-ESAT6-Rv1733c-Rv2626c-RpfB (SEQ ID NO:99 or SEQ IDNO:100); PPE51-Rv1733c-Rv2628c-RpfD (SEQ ID NO:64);PPE51-Rv1733c-Rv2628c-RpfB (SEQ ID NO:66); Rv3407-Rv1733c-Rv2626c-RpfB(SEQ ID NO:68); or Rv3407-Rv1733c-Rv2626c-RpfD (SEQ ID NO:70).
 2. Apharmaceutical composition comprising the fusion protein according toclaim 1 and a pharmaceutically acceptable carrier.
 3. The fusion proteinaccording to claim 1, wherein the fusion protein comprises an amino acidsequence at least 98% identical to an amino acid sequence ofAg85B-ESAT6-Rv1733c-Rv2626c-RpfD (SEQ ID NO:61 or SEQ ID NO:62).
 4. Afusion protein selected from the group consisting ofESAT6-Rv1733c-Rv2626c-RpfD (SEQ ID NO:53 or SEQ ID NO:54);ESAT6-Rv1733c-Rv2626c-RpfB (SEQ ID NO:56); RpfB-ESAT6-Rv1733c-Rv2626c(SEQ ID NO:58); Ag85B-ESAT6-Rv1733c-Rv2626c-RpfD (SEQ ID NO:61 or SEQ IDNO:62); Ag85B-ESAT6-Rv1733c-Rv2626c-RpfB (SEQ ID NO:99 or SEQ IDNO:100); PPE51-Rv1733c-Rv2628c-RpfD (SEQ ID NO:64);PPE51-Rv1733c-Rv2628c-RpfB (SEQ ID NO:66); Rv3407-Rv1733c-Rv2626c-RpfB(SEQ ID NO:68); or Rv3407-Rv1733c-Rv2626c-RpfD (SEQ ID NO:70).
 5. Thefusion protein according to claim 4, where in the fusion protein isAg85B-ESAT6-Rv1733c-Rv2626c-RpfD (SEQ ID NO:61 or SEQ ID NO:62).
 6. Amethod of eliciting an immune response against Mycobacteriumtuberculosis in a mammal comprising administering to the mammal animmunologically sufficient amount of the fusion protein according toclaim 1.